JP2003506014A - Compositions and methods for assaying subcellular conditions and processes using energy transfer - Google Patents

Abstract

(57) Abstract The present invention provides a subcellular compartment, such as an organelle, by an energy transfer technique that does not require a specific intermolecular affinity binding event between an energy transfer donor and an energy transfer acceptor molecule. Provided are compositions and methods for monitoring. Energy transfer methodologies, including fluorescence resonance energy transfer (FRET), provide methods for assaying cell membrane potential, including mitochondrial membrane potential. Diagnostic and drug screening assays are also provided.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates generally to biological assays for detecting physiological conditions within cells. More specifically, the present invention provides for monitoring molecular interactions in subcellular compartments based on energy transfer from a first compound (energy transfer donor) to a second compound (energy transfer acceptor). Regarding

BACKGROUND OF THE INVENTION Cells are the basic unit of life and contain various subcellular compartments, including, for example, organelles. Organelles are structural components of cells that are physically separated from components of other cells, typically by one or more membranes, which perform specialized cell functions. Organelles and other subcellular compartments vary in their composition and number, inter alia, in cells from different tissues, between normal and abnormal cells, and in cells from different species. Thus, organelles and other subcellular compartments, and macromolecules specifically associated with them, respectively, affect drugs that specifically affect specific tissues within an animal, and abnormal (affected) cells, respectively. It represents a novel target for the development of drugs that do not affect normal (healthy) cells, or drugs that specifically affect cells from unwanted species but not cells from desired species.

For example, members of the Bcl-2 family of proteins (discussed in more detail below) associate with the outer membrane of mitochondria and other cell membranes. Transfer of the Bcl-2 protein from one intracellular location to another (translocatio)
n) occurs during apoptosis. Apoptosis is a process by which some abnormal (eg, precancerous) cells are directed to undergo programmed cell death (PCD), thus removing their threat from the host organ. Means for monitoring the accumulation of Bcl-2 protein in various subcellular compartments, or its translocation from one intracellular location to another, is designed to affect apoptosis, and such agents in cells. Allows the identification of drugs designed to assay the effect of

As another example, cytoplasmic hybrids (cybrids) have been prepared that include the nucleus of one cell type and the organelles (mitochondria) from another cell type. Experiments with such cytoplasmic hybrids have demonstrated that the cellular defects associated with the affected cells are transmitted by the cytoplasmic element (mitochondria) from the affected cells to the cytoplasmic hybrid. Diseases that have been demonstrated to have cytoplasmic components in this manner include Alzheimer's disease and Parkinson's disease (Swerdlow et al., Ne.
urology 49: 918-925, 1997; Swerdlow et al., An.
nals of Neurology 40: 663-671, 1996). A means for monitoring intracellular processes during the formation of cytoplasmic hybrids, or intracellulars between cytoplasmic hybrids that have a common nuclear background but differ as a function of their donor cytoplasmic source as their mitochondrial source. Means for comparing processes allow the study of the mechanism of such processes and the identification of agents that affect such processes.

As a further example, a structure in which a bacterial cell is not present in a eukaryotic cell (eg,
It is possible to develop antibacterial agents by taking advantage of the fact that they contain cell walls) and by developing agents that specifically affect these structures. In contrast, the development of agents to treat diseases and disorders resulting from eukaryotic parasites in mammals, including humans, has been more difficult. This is because many features of the cells of such parasites have structural similarities to the homologous structures found in host cells; as a result, they are negative for the cellular constituents of such parasites. Any drug that affects A. pneumoniae is also probably due in part to the fact that it has a negative effect on similar components of eukaryotic host cells.

Accordingly, there is a need for methods and compositions that allow for rapid and detailed monitoring of processes within the subcellular compartment and associated macromolecules. In addition, there is a need for methods and compositions for identifying and screening agents that affect such processes in specific cases.

[0007] One object of the present invention is to provide methods and compositions for monitoring and assaying processes within subcellular compartments and associated macromolecules. If such a process is associated with a particular disease and / or disorder, the invention can be used in a predictive, diagnostic or prognostic modality.

[0008] Another object of the invention is to provide methods for screening and identifying agents that affect organelles and other subcellular compartments in a specific manner. When such agents are specific to unwanted abnormal cells or unwanted parasite cells, they are therapeutically or therapeutically effective against patients containing such unwanted cells. , Palliative, rehabilitative, preventive (preven
It is expected to have a positive, prophylactic or disease preventive effect.

The present invention fulfills these needs and fulfills these and other objectives. Other advantages of the invention will be apparent from the disclosure.

SUMMARY OF THE INVENTION The present invention relates in part to methods and compositions for monitoring cellular processes, conditions and molecules using energy transfer (ET) technology. Such ET-based methods and compositions further provide a means to screen and identify agents that alter (eg, increase or decrease) such processes, conditions and molecules in a statistically significant manner. To do. Accordingly, in one aspect, the present invention provides a method for assaying mitochondrial membrane potential, which comprises simultaneously or sequentially and in any order a sample containing one or more mitochondria. First and second not endogenous to mitochondria
Wherein each of the first and second energy transfer molecules is independent of each other and is the same sub-mitochondrial site or outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial membrane. Localized at an acceptably adjacent sub-mitochondrial site that is an interspace or mitochondrial matrix, and wherein the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule. Is a molecule,
Exciting the energy donor molecule to produce an excited energy donor molecule; and detecting a signal resulting from energy transfer from the first energy transfer molecule to the second energy transfer molecule. And
Where the concentration of at least one of these energy transfer molecules in the mitochondria changes as a function of membrane potential.

In a particular embodiment of this aspect of the invention, the excited energy donor molecule transfers energy to the energy acceptor molecule to produce an excited energy acceptor molecule, and the detected signal. From the energy released by the excited energy acceptor molecule. In certain embodiments, energy transfer from the first energy transfer molecule to the second energy transfer molecule results in a decrease in the detectable signal. In certain further embodiments, the method comprises:
Contacting the mitochondria with an agent that induces mitochondrial membrane potential dissipation. In certain other embodiments, the agent that induces the consumption of mitochondrial membrane potential is an ionophore. In certain additional embodiments, the method comprises contacting the mitochondria with an agent that induces a collapse of mitochondrial membrane potential. In another embodiment, the agent that induces a sudden drop in mitochondrial membrane potential is CCC.
P or FCCP. In certain embodiments, the sample is washed prior to the step of detecting the signal, and in another embodiment the detected signal is compared to the reference signal. In certain further embodiments, the reference signal is generated by an index of cell number, an index of mitochondrial mass, an index of cellular protein, an index of cellular DNA, an index of mitochondrial DNA, an index of mitochondrial protein, and an index of fluid volume. .

In another embodiment of the invention, the sample comprises one or more mitochondria present in at least one cell and the detected signal is compared to a reference signal. In certain further embodiments, the reference signal is generated from an intracellular site that can be the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space, mitochondrial matrix, cytoplasm, nucleus, nuclear membrane or plasma membrane. In another embodiment, the reference signal is produced from the extracellular medium. In another embodiment, the mitochondria are present in at least one cell during at least one step, and in certain further embodiments, the cell is
An organism, a cultured cell, a cytoplasmic hybrid cell, a plant cell or an animal cell. In certain other embodiments, the cells are in a biological sample from a multicellular organism, in some embodiments the cells are plant cells, and in other embodiments, animal cells. In some embodiments, the animal is a mammal, and in some embodiments, the mammal is a human. In a further embodiment, the human has, is suspected of or at risk of having, a disease or disorder associated with organelle dysfunction, and in certain further embodiments, the dysfunction is a mitochondrial function. Deficiency, and in certain other embodiments, lysosomal dysfunction.

In another embodiment of this aspect of the invention, the first energy transfer molecule is localized to the mitochondrial site of the mitochondrial matrix or the inner mitochondrial membrane, and the second energy transfer molecule is the mitochondrial matrix. Alternatively, it is localized in the sub-mitochondrial region, which is the inner mitochondrial membrane. In one embodiment, the concentration of the first energy transfer molecule at this submitochondrial site does not change as a function of membrane potential and the concentration of the second energy transfer molecule at the mitochondrial matrix decreases as a function of membrane potential. To do. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 373 nm to about 390 nm and an emission maximum at a wavelength of about 400 nm to about 500 nm; and a second energy transfer molecule. Has an excitation maximum at a wavelength of about 400 nm to about 500 nm. In a further embodiment, the first energy transfer molecule is a fusion protein, wherein the fusion protein is Phe-64, Ser-6.
5, a blue-shifting green fluorescent protein polypeptide having a mutation in at least one of Tyr-66, Val-68 and Tyr-145, and the fusion protein to a mitochondrial site that is the mitochondrial matrix or inner mitochondrial membrane. The second energy transfer molecule comprises a polypeptide sequence that localizes; and the second energy transfer molecule is DASPEI, DASPMI, 4-Di-1-ASP, 2-D.
i-1-ASP, DiOC ₇ (3), DiOC ₆ (3), JC-1 or SYTO
(Registered trademark) 18 yeast mitochondrial stain. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 425 nm to about 440 nm and an emission maximum at a wavelength of about 450 nm to about 535 nm; and a second energy transfer molecule. Has an excitation maximum at a wavelength of about 450 nm to about 530 nm.

In another embodiment, the first energy transfer molecule is a fusion protein,
Here, this fusion protein is Phe-64, Ser-65, Tyr-66, A
a blue-green shift protein polypeptide having a mutation in at least one of sn-146, Met-153, Val-163 and Asn-212, and the fusion protein selected from the group consisting of mitochondrial matrix and inner mitochondrial membrane The second energy transfer molecule is DASPEI, 2-Di-1-A.
SP, DiOC ₆ (3), SYTO® 18 yeast mitochondrial stain, Rhodamine 6G, JC-1, NBD C6-ceramide or NBD C6-sphingomyelin. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 470 nm to about 500 nm, and about 505 nm to.
The emission maximum at a wavelength of about 565 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of about 505 nm to about 565 nm.

In yet another embodiment, the first energy transfer molecule is nonyl acridine orange, Mito Tracker® Green FM, Mito.
Fluor ^™ Green or a fusion protein, wherein the fusion protein is a wild-type green fluorescent protein polypeptide, Phe-64, Ser-6.
5, 1 of Tyr-66, Gln-69, Ser-72 and Thr-203
Red-shifting green fluorescent protein polypeptide having a mutation in one or more, or yellow having a mutation in one or more of Phe-64, Ser-65, Tyr-66, Gln-69, Ser-72 and Thr-203. A green fluorescent protein polypeptide, which is a biased green fluorescent protein polypeptide, as well as a polypeptide sequence that localizes the fusion protein to a sub-mitochondrial site of the mitochondrial matrix or inner mitochondrial membrane; and a second energy transfer molecule Is rhodamine 123, JC-1, tetrabromorhodamine 123,
Rhodamine 6G, TMRM, TMRE, tetramethylrhodamine (tetram)
Ethylrosamine) or Rhodamine B. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 545 to about 560 nm and an emission maximum at a wavelength of about 565 to about 625 nm; and a second energy transfer molecule. Has an excitation maximum at a wavelength of about 565 to about 625 nm. In a further embodiment, the first energy transfer molecule is Mito Tracke.
r be a (R) Orange CMTMRos; and second energy transfer molecule is a DiOC ₂ (5). In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 495 to about 510 nm and about 51
It has an emission maximum at a wavelength of 0 to about 570 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of about 510 to about 560 nm. In another embodiment,
The first energy transfer molecule is a fusion protein, where the fusion protein is the FLASH protein sequence or Ser-65, Tyr-66, Ser-.
72 and a yellow-shifted green fluorescent protein polypeptide sequence having a mutation in one or more of Thr-203, and the fusion protein localized to the mitochondrial matrix and the sub-mitochondrial site of the inner mitochondrial membrane. The second energy transfer molecule comprises JC-1, tetrabromorhodamine 123, rhodamine 6G, T.
MRM, TMRE, tetramethylrhodamine, rhodamine B and 4-dimethylaminotetramethylrosamine.

In another embodiment of this aspect of the invention, the relative amount of signal produced by energy transfer is detected. In certain other embodiments, the signal is detected over a period of time, and the rate of change in signal level is determined, and in certain other embodiments, the signal is detected over a period of time, and Integrated. In another embodiment, the membrane potential comprises a potential, a pH potential, or both. In one embodiment, the first and second energy transfer molecules are localized within about 10 angstroms to about 100 angstroms of each other, and in another embodiment, they are about 10 angstroms of each other.
Localized within angstroms to about 50 angstroms, and in another embodiment, they are located within about 20 angstroms to about 50 angstroms of each other. In certain embodiments, this signal is generated by fluorescence resonance energy transfer.

In another aspect, the invention provides a method for identifying an agent that alters mitochondrial membrane potential, which method comprises the steps of: in the presence and absence of a candidate agent. Contacting a sample containing one or more mitochondria with each of a first energy transfer molecule and a second energy transfer molecule that is not endogenous to the mitochondria, either simultaneously or sequentially and in any order. So, the first energy transfer molecule and the second energy transfer molecule are
Each independently of the other, localized to the same sub-mitochondrial site or an admissibly adjacent sub-mitochondrial site, which is the mitochondrial outer membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix, and The first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to generate an excited energy donor Generating a molecule; detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria is: Changes as a function of membrane potential,
And comparing the signal generated in the absence of the candidate drug with the signal generated in the presence of the candidate drug, and from there identifying the drug that alters the mitochondrial membrane potential.

In another aspect, the invention provides a method for identifying a regulator of a drug that alters mitochondrial membrane potential, which method comprises the steps of: in the presence and absence of a candidate regulator. Below, an agent that alters the mitochondrial membrane potential (including agents as identified according to the methods provided herein above) and a sample containing one or more mitochondria is not endogenous to this mitochondria. A step of contacting each of the first energy transfer molecule and the second energy transfer molecule simultaneously or sequentially and in any order, the first energy transfer molecule and the second energy transfer molecule comprising: Each sub-mitochondria, independently of each other, in the same sub-mitochondrial site or in an admissibly adjacent region Is localized to the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix, and the first energy transfer molecule is the energy donor molecule and the second energy transfer molecule is The energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule; from the first energy transfer molecule to the second energy transfer molecule Detecting the signal generated by the energy transfer of the protein, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential; and is generated in the absence of a candidate regulator. The generated signal in the presence of the candidate regulator. Step comparing Null, and from there, identifying a regulator of an agent to alter the mitochondrial membrane potential. In one embodiment, the regulator is an agonist of a drug that alters mitochondrial potential, and in another embodiment, the regulator is an antagonist of a drug that alters mitochondrial potential. In another embodiment, the agent that alters the mitochondrial membrane potential is an apoptoge.
n). In another embodiment, the agent that alters mitochondrial membrane potential is thapsigargin, an ionophore, an excitatory amino acid or a derivative thereof. In certain further embodiments, the ionophore is ionomycin or A
23187. In certain other embodiments, the excitatory amino acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate.

In another aspect, the invention substantially comprises mitochondrial membrane potential in mitochondria from a first biological source and mitochondrial membrane potential in mitochondria from a second biological source. A method for identifying a drug that is preferentially altered without alteration is provided, which method comprises the following steps: a method comprising one or more mitochondria in the presence and absence of a candidate agent. One biological sample and a second biological sample are each loaded with a first energy transfer molecule and a second energy transfer molecule that are not endogenous to this mitochondria, either simultaneously or sequentially. Contacting in sequence, the first sample being from a first biological source and the second sample being Not derived from the first biological source, the first energy transfer molecule and the second energy transfer molecule are each independently of the other at the same sub-mitochondrial site or at an acceptably adjacent sub-mitochondrial site. Localized, this site is the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix, and the first energy transfer molecule is the energy donor molecule and the second energy transfer molecule. The molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule in the presence of each of the first sample and the second sample; From this first energy transfer molecule in the presence of each of the first and second samples Detecting the signal generated by energy transfer to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential; The signal produced in the presence of each of the first sample and the second sample in the absence of the first sample and the second sample in the presence of the candidate agent is
Comparing the signal generated in the presence of each of the samples of, and from there identifying agents that preferentially alter the mitochondrial membrane potential.

In one embodiment, the first and second biological sources are distinct biological species, and in another embodiment, the first biological source is diseased. A mammal suspected of having, diagnosed as having, or predisposing to have the disease, and the second biological source is not suspected of having the disease , A mammal that has not been diagnosed with the disease or is not predisposed to have the disease. In a further embodiment,
The first biological source is human and the second biological source is human. In another embodiment, the disease is Alzheimer's disease, Parkinson's disease or type II diabetes.

In another aspect, the invention provides for mitochondrial membrane potential in mitochondria from a first biological sample without substantially altering mitochondrial membrane potential in mitochondria from a second biological sample. , Provides a method for identifying a drug that alters preferentially, the method comprising the steps of: a first biology comprising one or more mitochondria in the presence and absence of a candidate drug. A first energy transfer molecule and a second energy transfer molecule each of which is not endogenous to this mitochondria, either simultaneously or sequentially and in any order. Contacting, wherein the first sample is from the first tissue and the second sample is the first tissue. Is derived from a second tissue different from the tissue, the first energy transfer molecule and the second energy transfer molecule being independent of each other at the same submitochondrial site or at an acceptably adjacent submitochondrial site Localized, this site is the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix, and the first energy transfer molecule is the energy donor molecule and the second energy transfer molecule. The molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule in the presence of each of the first sample and the second sample; In the presence of each of the first and second samples, this first energy transfer molecule Detecting a signal generated by energy transfer to a second energy transfer molecule, wherein the concentration of at least one energy transfer molecule in the mitochondria changes as a function of membrane potential; Signal generated in the presence of each of the first sample and the second sample in the absence of H2O2 was generated in the presence of each of the first sample and the second sample in the presence of the candidate agent Comparing to the signal and from there identifying the agent that preferentially alters the mitochondrial membrane potential. In one embodiment, the first tissue and the second tissue are from the same subject, while in another embodiment, the first tissue and the second tissue are each from the same species of subject. Is. In another embodiment, the first tissue and the second tissue are from subjects of different species.

Yet another aspect of the invention is to provide a method of detecting the fusion of a first mitochondria and a second mitochondria, the method comprising the steps of: Contacting a first sample containing with a first energy transfer molecule that is not endogenous to this mitochondria; a second sample containing one or more mitochondria, a second sample that is not endogenous to this mitochondria Contacting the first energy transfer molecule and the second energy transfer molecule, each independently of the other, at the same sub-mitochondrial site or an acceptably adjacent sub-mitochondrial site. Localized,
This site is the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix, and the first energy transfer molecule is the energy donor molecule and the second energy transfer molecule is the energy transfer molecule. An acceptor molecule; contacting a first sample with a second sample under conditions and for a time sufficient to allow mitochondrial fusion; exciting this energy donor molecule to provide an excited energy donation Generating a somatic molecule; detecting a signal generated by the energy transfer from the first energy transfer molecule to the second energy transfer molecule, and from there the fusion of the first and second mitochondria The process of measuring.

In another aspect, the invention provides a method of identifying an agent that alters mitochondrial fusion, the method comprising the steps of: a first sample comprising one or more mitochondria, Contacting a first energy transfer molecule that is not endogenous to this mitochondria; contacting a second sample containing one or more mitochondria with a second energy transfer molecule that is not endogenous to this mitochondria Wherein the first energy transfer molecule and the second energy transfer molecule are each independently of one another located at the same sub-mitochondrial site or at an acceptably adjacent sub-mitochondrial site, which site is , Outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix And the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; mitochondrial fusion in the presence and absence of a candidate agent. Contacting a first sample with a second sample under conditions and for a time sufficient to allow; allowing the energy donor molecule to produce an excited energy donor molecule; Detecting a signal generated by energy transfer from one energy transfer molecule to a second energy transfer molecule; and a signal detected in the absence of a candidate agent and a signal detected in the presence of a candidate agent Comparing and, from there, identifying agents that alter mitochondrial fusion. In certain embodiments, the agent increases mitochondrial membrane potential, in certain other embodiments, the agent consumes mitochondrial membrane potential, and in certain other embodiments, the agent increases mitochondrial membrane potential. The potential is plunged, and in certain embodiments, the agent is at least 1 on either side of the cell membrane.
Change the equilibrium distribution of two ionic species. In a further embodiment, the ionic species is Ca ²⁺ and the cell membrane is a mitochondrial membrane. In certain embodiments, the agent that causes a drop in mitochondrial membrane potential is an apoptogen,
And in certain other embodiments, the agent that causes a drop in mitochondrial membrane potential interacts with an adenine nucleotide translocator, and in certain other embodiments, the agent that causes a drop in mitochondrial membrane potential is atractyloside, carboxy. It is atractyloside, boncrecic acid, or isobombrecic acid.

In another aspect, the invention provides a reagent for measuring mitochondrial ΔΨ, comprising a FRET donor molecule and a FRET acceptor molecule, wherein accumulation of at least one of those molecules in mitochondria is It is dependent on ΔΨ and the accumulation of the other molecule in mitochondria is independent of ΔΨ. In one embodiment, molecules that accumulate in mitochondria independent of ΔΨ are NAO
, MitoTracker (registered trademark), Green FM, MitoFluo
r ^™ , DAPI, or a fusion protein, wherein the fusion protein is a red-shifted green fluorescent protein polypeptide, a yellow-shifted green fluorescent protein polypeptide or a “FLASH” polypeptide and the mitochondrial matrix or It contains a polypeptide sequence that localizes this fusion protein to the membrane. In certain other embodiments, the molecules that accumulate in mitochondria in a ΔΨ-dependent manner are TMRM, TMRE, rhodamine 123, ethidium bromide, 4
-Di-1-ASP, 2-Di-1-ASP or DASPEI. In certain embodiments, the invention also provides the described reagents and mitochondrial ΔΨ.
A kit is provided which comprises auxiliary reagents for measuring

Another aspect of the invention is to provide a method for assaying cell membrane potential, the method comprising the steps of: to a sample containing at least one cell membrane, to this sample. Contacting each of the first and second non-endogenous energy transfer molecules simultaneously or sequentially and in any order, the first energy transfer molecules and the second energy transfer molecules The energy transfer molecules are independent of each other, and at least one energy transfer molecule is
Localized at the same or receptively adjacent membrane site to localize at the cell membrane forming the subcellular compartment, and the first energy transfer molecule is an energy donor molecule, and The second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule; and from the first energy transfer molecule to the second energy transfer molecule. A step of detecting a signal generated by energy transfer to an energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules at this membrane site is
A process that changes as a function of membrane potential. In one embodiment, the first energy transfer molecule is localized at a first membrane site, which is a mitochondrial membrane, endoplasmic reticulum membrane, Golgi membrane, lysosomal membrane or plasma membrane, and a second energy transfer molecule. Is localized to the same or receptively adjacent membrane sites, which are mitochondrial, endoplasmic reticulum, Golgi, lysosomal or plasma membranes. In another embodiment, the concentration of the first energy transfer molecule in the first membrane site does not change as a function of the membrane potential, and the concentration of the second energy transfer molecule in the membrane site is Decreases as a function of.

In one embodiment, the first energy transfer molecule is about 373 nm to about 3 nm.
It has an excitation maximum at a wavelength of 90 nm and an emission maximum at a wavelength of about 400 nm to about 500 nm; and the second energy transfer molecule is about 400 nm to about 500 nm.
It has an excitation maximum at a wavelength of nm. In a further embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 425 nm to about 440 nm, and about 4
The emission maximum at a wavelength of 50 nm to about 535 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of about 450 nm to about 530 nm. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of about 470 nm to about 500 nm and an emission maximum at a wavelength of about 505 nm to about 565 nm;
Then, the second energy transfer molecule has an excitation maximum at a wavelength of about 505 nm to about 565 nm. In another embodiment, the first energy transfer molecule is about 545
Has an excitation maximum at a wavelength of about 560 nm and an emission maximum at a wavelength of about 565 to about 625 nm; and the second energy transfer molecule is about 565 to about 625n.
It has an excitation maximum at a wavelength of m.

In yet another aspect, the invention provides a method for identifying an agent that alters cell membrane potential, the method comprising the steps of: in the presence and absence of a candidate agent, Contacting a sample containing one or more cell membranes simultaneously with each of the first energy transfer molecule and the second energy transfer molecule that are not endogenous to the sample, or sequentially in either order. And wherein each of the first energy transfer molecule and the second energy transfer molecule localizes independently of one another to the same or acceptably adjacent membrane site, such that at least One energy transfer molecule localizes to the cell membrane forming the subcellular fraction, and the first energy transfer molecule is the energy donor molecule. And a second energy transfer molecule is an energy acceptor molecule; a step of exciting the energy donor molecule to produce an excited energy donor molecule; Detecting a signal generated by energy transfer to an energy transfer molecule, wherein the concentration of at least one energy transfer molecule in the subcellular fraction changes as a function of membrane potential; and a candidate agent. Comparing the signal generated in the absence of the drug with the signal generated in the presence of the candidate drug and then identifying the drug that alters the cell membrane potential.

Another aspect of the invention provides a method for identifying a regulator of an agent that alters cell membrane potential, the method comprising the steps of: in the presence and absence of a candidate regulator, A sample comprising an agent that alters a cell membrane potential (which may be an agent identified according to the method just described) and one or more cell membranes is provided with a first energy transfer molecule that is not endogenous to the sample and a second.
Of each of the first energy transfer molecules and the second energy transfer molecules independently of each other and the same. Localized to a membrane site or an acceptably adjacent membrane site such that at least one of the energy transfer molecules localizes to a cell membrane forming a subcellular fraction, and the first energy transfer molecule is an energy donor. A molecule and the second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule; A step of detecting a signal generated by energy transfer to a second energy transfer molecule, wherein the energy in the subcellular fraction is detected. Comparing the signal generated in the absence of the candidate regulator with the signal generated in the presence of the candidate regulator, and then at least one concentration of the gee transfer molecule changes as a function of the membrane potential; Identifying regulators of drugs that alter cell membrane potential.

Another aspect of the present invention prefers the cell membrane potential at the membrane from the first biological source without substantially altering the cell membrane potential at the membrane from the second biological source. A method for identifying a chemically altered agent, which method comprises the following steps: a first biological sample comprising one or more cell membranes in the presence and absence of a candidate agent. And contacting each of the second biological sample with each of the first energy transfer molecule and the second energy transfer molecule that are not endogenous to the sample, either simultaneously or sequentially in either order. A step, wherein the first sample is from a first biological source and the second sample is a second biological source different from the first biological source. Is the origin and the first energy transfer Each of the molecule and the second energy transfer molecule, independently of each other, localizes to the same membrane site or an acceptably adjacent membrane site such that at least one of the energy transfer molecules forms a subcellular fraction. Localized to the cell membrane, and the first
The energy transfer molecule is an energy donor molecule, and the second energy transfer molecule is an energy acceptor molecule, the energy donor molecule being in the presence of each of the first sample and the second sample. Exciting to produce an excited energy donor molecule; by energy transfer from the first energy transfer molecule to the second energy transfer molecule in the presence of each of the first sample and the second sample Detecting the signal generated, wherein the concentration of at least one energy transfer molecule in the subcellular fraction changes as a function of membrane potential; and in the absence of the candidate agent, First sample and second
Signal generated in the presence of each of the samples of
Of the sample and the second sample in the presence of each, and then identify agents that preferentially alter the cell membrane potential.

In another aspect, the present invention preferentially favors cell membrane potential in a membrane from a first biological sample without substantially altering cell membrane potential in a membrane from a second biological sample. To provide a method for identifying a drug that alters
The following steps are included: each of the first biological sample and the second biological sample containing one or more cell membranes in the presence and absence of the candidate agent, either simultaneously or in any order. Continuously contacting each of a first energy transfer molecule and a second energy transfer molecule that is not endogenous to the sample,
Wherein the first sample is from the first tissue and the second sample is the first
Is derived from a second tissue different from that of the first energy transfer molecule and the second energy transfer molecule.
Of each of the energy transfer molecules of the L., independently of one another, are localized at the same or receptively adjacent membrane sites, such that at least one of the energy transfer molecules is at the cell membrane forming the subcellular fraction. Localized, and the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule, Step: in the presence of each of the first sample and the second sample A second energy from the first energy transfer molecule in the presence of each of the first sample and the second sample, the step of exciting the energy donor molecule to produce an excited energy donor molecule. Detecting a signal generated by energy transfer to a transfer molecule, wherein at least the energy transfer molecule in the subcellular fraction is The two concentrations vary as a function of membrane potential, and the signal generated in the presence of each of the first sample and the second sample in the absence of the candidate agent is measured in the presence of the candidate agent. , Comparing the signal generated in the presence of each of the first and second samples, and then identifying agents that preferentially alter the cell membrane potential.

In yet another aspect, the invention provides a method for detecting a particular type of cell in a sample, the method comprising the steps of: a sample containing one or more mitochondria. Contacting each of the first energy transfer molecule and the second energy transfer molecule that are not endogenous to this mitochondria, simultaneously, or sequentially in either order, wherein the first energy transfer Each of the molecule and the second energy transfer molecule, independently of each other, localizes to the same subcellular site or an acceptably adjacent subcellular site, and the first energy transfer molecule is an energy donor cell, And the second energy transfer molecule is an energy acceptor molecule, the step; exciting the energy donor molecule to provide the excited energy donation Producing a body molecule; and detecting a signal produced by energy transfer from a first energy transfer molecule to a second energy transfer molecule, wherein at least one of the energy transfer molecules is , Preferentially accumulating in specific types of cells, where the signal correlates with the presence of specific types of cells in the sample. In one embodiment, the method further comprises comparing the signal generated in the sample with the signal generated from control cells lacking a particular type of cell. In another embodiment, the particular type of cell is a cancer cell.

In another aspect, the invention provides a method for identifying a ΔΨ _m stabilizing agent, which method comprises the following steps: in the presence and absence of a candidate ΔΨ _m stabilizing agent. And a sample containing an agent that alters ΔΨ _m and one or more mitochondria, simultaneously or sequentially in either order, a first energy transfer molecule and a second energy transfer molecule that are not endogenous to this mitochondria. Wherein each of the first energy transfer molecule and the second energy transfer molecule is independently of one another localized at the same sub-mitochondrial site or an acceptably adjacent sub-mitochondrial site. This site may be the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space or mitochondrial matrix. And the first energy transfer molecule is an energy donor molecule,
And the second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce the excited energy donor molecule;
Detecting a signal generated by energy transfer from a first energy transfer molecule to a second energy transfer molecule, wherein at least one concentration of the energy transfer molecule in the mitochondria is at a membrane potential. varies as a function; and a signal generated in the absence of the candidate [Delta] [Psi] _m stabilization agent, relative to the signal generated by the presence of the candidate [Delta] [Psi] _m stabilization agent, and then, [Delta] [Psi] _m stabilization The process of identifying a drug. In one embodiment, mitochondria are contained intracellularly, and in a further embodiment, the agent that alters ΔΨ _m is an agent that increases levels of cytosolic Ca 2+. In another embodiment, the agent that increases levels of cytosolic Ca2 + is a calcium ionophore or thapsigargin. In another embodiment, the cells contain one or more types of glutamate receptors. In another further embodiment, the agent that increases levels of cytosolic Ca2 + is an excitatory amino acid or derivative thereof. In another further embodiment, the excitatory amino acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate.
In another embodiment, the invention provides a ΔΨ _m stabilizing agent identified according to the method just described. In another embodiment, the present invention provides a method of treating stroke, comprising administering a ΔΨ _m stabilizing agent to a patient in need thereof.

These and other aspects of the invention will be apparent with reference to the following detailed description and accompanying drawings. All references disclosed herein are incorporated herein by reference as if each were individually incorporated in their entirety.

(Symbols and Abbreviations) A description of the terminology and abbreviations is listed in Table 1. Unless otherwise indicated, the symbols for nucleotides and amino acids are as set forth in 37 CFR §1.821.

[0035]

[Table 1] ^* Abbreviations for suppliers: “Calbiochem”, Calbioche
m, Inc. , La Jolla, CA; "MP", Molecular Pr.
obes, Inc. , Eugene, OR; "Biomol", Biomol
Research: Laboratories, Inc. , Plymouth
Meeting, MA; "Mol. Dev.", Molecular Dev.
ices, Sunnyvale, CA; "Aurora", Aurora Bi
oSciences Corp. , San Diego, CA; "Clonte
ch ”, CLONTECH Laboratories Inc. , Palo
Alto, CA; "Sigma", Sigma Chemical Co. , S
t. Louis, MO; RBI, Research Biochemical
s International, Natick, MA.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, in part, to the use of intermolecular energy transfer to monitor intracellular and intracellular conditions. In particular, the invention uses donor-acceptor pairs of energy transfer molecules in which such intracellular and organelle states need not undergo specific intermolecular recognition events (eg, affinity binding interactions). And comes from the unexpected observation that it can be evaluated. More precisely, according to the present disclosure, in order to produce a detectable signal under certain naturally occurring or artificially induced intracellular and / or organelle physiological conditions. Appropriately paired energy transfer donor and acceptor molecules that accumulate at acceptably contiguous sites (provided herein) can be selected.

By way of background, energy transfer (ET) results from resonant interactions between two molecules: an “donor” molecule that donates energy and an “acceptor” molecule that accepts energy. Energy transfer can occur in the following cases: (1) if the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor, and (2) the donor and acceptor are at a specific distance from each other (eg, (Less than about 10 nm). Energy transfer efficiency is greatly affected by the proximity of donor and acceptor and decreases as the sixth power of distance. Thus, ET measurements strongly reflect the proximity of acceptor and donor compounds, and changes in ET change in proximity of these compounds (eg, to the association or dissociation of donor and acceptor). Like)
Reflect in high sensitivity.

According to the present invention, both energy transfer molecules (ET donor molecule and ET acceptor molecule) are contacted with them in a sample as provided herein (non-limiting example). Include cells, organelles (eg, mitochondria),
Or a subcellular compartment or a suborganelle compartment). The donor and acceptor compounds may be co-localized in the subcellular compartments in such a way as to achieve sufficient proximity to one another for certain types of energy transfer to occur. In certain aspects of the invention, such co-localization may depend on, or be disrupted by, intracellular processes or responses to chemical agents. For example, such a process or response can lead to an increase or decrease in energy transfer, which can be detected by detecting the signal, respectively. Thus, for example, detecting the extent or rate of energy transfer between an ET donor molecule and an ET acceptor molecule can provide a method of assaying a given intracellular process or response at the appropriate location. In certain preferred embodiments, the invention provides methods for assaying cell membrane potential. And in certain other preferred embodiments, the invention provides methods for assaying mitochondrial membrane potential.

Accordingly, it is an aspect of the present invention to provide a method of assaying cell membrane potential, which method, where appropriate, comprises a sample containing one or more cell membranes, ET
Contacting the donor and ET acceptor molecules, exciting the ET donor to produce an excited ET donor molecule, and a signal generated by energy transfer from the ET donor to the ET acceptor Depending on the step of detecting. The sample may be contacted simultaneously with the ET donor and ET acceptor, depending on the particular donor and acceptor used, or contacted with the ET donor and ET acceptor sequentially and in any order. Can be done. If desired, the sample may be washed under suitable conditions prior to the step of detecting the signal, eg, to improve the sensitivity of detecting the signal. One of ordinary skill in the art can readily determine the manner in which the sample is contacted in light of the properties of the sample and ET molecule selected, and in light of the teachings provided herein. Also provided herein, the methods of the invention are any suitable ET donor molecule and E capable of functioning as a donor-acceptor pair.
T receptor molecules can be used. As discussed in more detail below, the methods of the invention can be used to identify agents that alter cell membrane potential, or to identify molecules that are regulators of such agents.

In certain preferred embodiments, the invention relates to a method of assaying mitochondrial membrane potential, wherein neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and here: The ET donor and the ET acceptor, independently of each other, each have the same submitochondrial site (submitochondr).
ial site) or, as provided herein, localized to an admissibly adjacent sub-mitochondrial site.

Optionally, in a preferred embodiment, both the ET donor molecule and the ET acceptor molecule emit a detectable signal in the form of light when excited by excitation light of the appropriate wavelength. It may be a light emitting molecule such as a fluorescent molecule, a phosphorescent molecule or a chemiluminescent molecule. Preferred ET Donor-E that can be Used According to the Invention
The combination of T acceptors is a combination of a fluorescent donor and a fluorescent acceptor or a phosphorescent acceptor, or a combination of a phosphorescent donor and a phosphorescent acceptor or a fluorescent acceptor. "Fluorescence" is the absorption of radiation at one wavelength ("excitation"), followed by:
Disappears almost instantly when the incident radiation is stopped, usually at different wavelengths,
Luminescence (luminescence) caused by almost immediate re-emission ("luminescence"). At the molecular level, fluorescence occurs as a particular compound (known as a fluorophore) is transferred by light energy from its ground state to a higher excited state; typically, it differs as this molecule returns to its ground state. It emits light at a wavelength. "Phosphorescence", by contrast, refers to luminescence that is caused by the absorption of radiation at one wavelength, followed by delayed re-emission that occurs at a different wavelength, and which persists for a significant period of time after the incident radiation has stopped. . "Chemiluminescence" refers to the luminescence that results from a chemical reaction, and "bioluminescence" refers to the emission of light from an organism or cells, organelles or extracts derived therefrom.

In certain preferred embodiments, the detectable signal produced by the energy transfer between the ET donor molecule and the ET acceptor molecule is fluorescence resonance energy transfer (F
RET). FRET occurs intramolecularly or between two different types of molecules when energy from an excited donor fluorophore is transferred directly to the acceptor fluorophore (for a review see Wu et al., Analyti).
cal Biochem. 218: 1-13, 1994). Generally, an excited fluorophore (eg, ET donor molecule) to an absorber (eg, E
Energy transfer to the T acceptor molecule) is measured by: (1) measuring the spectrum (including the change in spectrum) of fluorescence from the energy donor molecule and the energy acceptor molecule; Measuring the rate at which the intensity of the fluorescence intensity of the donor molecule decreases after pulsed laser excitation (ie fluorescence lifetime); or (3) the decrease in fluorescence intensity from the energy donor compound (ie indirect measurement of FRET). ), Or measuring the increase in fluorescence intensity from the energy acceptor compound (ie, a direct measurement of FRET). Direct measurement of energy transfer involves monitoring the signal from excited energy acceptor molecules, which increases as the ET compounds approach each other. On the other hand, indirect measurement of energy transfer involves monitoring the signal from the excited ET donor molecule, which decreases (ie, is quenched) as the compound becomes proximal (FIG. 1).

Specific intermolecular and specific intramolecular recognition events that bring fluorophores of the ET donor and ET acceptor into close proximity to each other (eg, association and dissociation events such as affinity and binding interactions). For monitoring specific intermolecular and / or intramolecular interactions, including
The use of is known in the art. When measuring such intermolecular interactions,
The ET donor fluorophore and ET acceptor fluorophore are typically located on two different molecules that are known to be or are considered to be in close association with each other. On the other hand, when intramolecular interactions are measured, the ET donor fluorophore and ET acceptor fluorophore are on the same molecule.

In contrast to such known uses of FRET methodology, where the ET donor fluorophore and ET acceptor fluorophore are brought into close proximity to each other through known specific molecular interactions, the present invention provides a common Subcellular compartment of (
Energy between an ET donor fluorophore and an ET acceptor fluorophore brought into proximity to each other by selective enrichment or accumulation in, for example, organelles, sub-organelle sites or locations in other sub-cells). Based on the unexpected observation that migration can occur. As a result, the invention can be used to monitor various conditions or processes within or associated with such subcellular compartments.

As provided herein, contemplated uses of the invention include, but are not limited to, the following: (i) Conditions and processes within the subcellular compartment. (Ii) monitoring the interaction between pairs of macromolecules found in or associated with such subcellular compartments, (iii) subcellular Identifying agents that affect compartments and / or intracellular processes in a species-specific manner, and (iv) subcellular compartments and / or intracellular processes in mammals, including humans, and other animals, and in plants. Identifying agents that affect in a manner that treats diseases and disorders. Each of these applications is described in more detail below.

[0046] Typically, the present invention relates, in part, to a method for assaying a sample, which in a preferred embodiment is a biological sample, and in a particularly preferred embodiment an organism containing one or more mitochondria. It is a scientific sample. In other preferred embodiments, the biological sample comprises one or more cell membranes (plasma membrane and intracellular membrane bound compartments (eg, endosomes, lysosomes, peroxisomes, mitochondria, chloroplasts, endocytic vesicles and secretory vesicles). , ER-Golgi component, organelle, etc.)). The biological sample can be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or biological source. The subject or biological source may be a human or non-human animal, a plant, a unicellular or multicellular organism, a primary cell culture or a culture adapted cell line (a gene that may include chromosomally integrated or episomal recombinant nucleic acid sequences. Engineered cell lines, immortalized cell lines or immortalizable cell lines, somatic cell hybrids or cytoplasmic hybrid "cybrid" cell lines, differentiated cell lines or differentiable cell lines,
(Including but not limited to transformed cell lines).

In certain embodiments of the invention it may be preferred to use whole intact cells, in other particular embodiments the use of permeabilized cells may be preferred. Permeabilized cells are cells that have been treated in such a way as to result in a partial or complete loss of selective permeability of the plasma membrane. As a first example, it may be preferable to permeabilize cells in a manner that allows calcium cations in the extracellular environment to diffuse into the permeabilized cells and contact the mitochondria. Thus, in this example, permeabilization serves as an alternative to the use of calcium ionophores. Second
As an example, certain detectably labeled molecules (eg, certain ET donor molecules and / or ET acceptor molecules provided herein) permeate the plasma membrane at moderate rates. Or, they can penetrate to a limited extent unless their entry into the cytosol is facilitated in any way. Cell permeabilization is one way in which the cytosolic entry of such ET molecules can be facilitated. As a third example, some candidate agents tested according to this method are capable of permeabilizing the plasma membrane at moderate rates, or unless their entry into the cytosol is facilitated in any way. It can be transmitted to the extent that it is done. Cell permeabilization is one way in which such candidate agents can be facilitated to enter the cytosolic space. Active agents identified under these conditions may then be chemically modified to enhance their uptake by whole cells; active agents so modified may be used for drug development. It is expected to serve as lead compounds, and in some cases, themselves can be used as drugs or drug candidates.

The person skilled in the art is familiar with methods for permeabilizing cells, for example, but by way of example and not limitation, by the following: surfactants.
, Through the use of detergents, detergents, phospholipids, phospholipid binding proteins, enzymes, viral membrane fusion proteins, etc .; by exposure to certain bacterial toxins (eg, α-hemolysin); hemolysin (eg, saponin (this) Are also nonionic detergents like digitonin)); through the use of osmotically active agents; by the use of chemical cross-linking agents; by physicochemical methods including electroporation, or for example, By other permeabilization methodologies involving physical manipulations such as electroporation. Those of skill in the art are familiar with methods for permeabilizing cells and, as provided herein, involve undue experimentation with the most suitable permeabilizing agent for use according to the invention. Can be easily determined without. Factors associated with this determination include, but are not limited to, the toxicity of the permeabilizing agent to particular cells, the molecular size of the molecule sought to enter the cell through the use of permeabilization (eg Schulz. , Methods Enzymol
． 192: 280-300, 1990).

Thus, for example, cells may be transfected with any of a variety of known techniques (permeabilizing agents (eg, bacterial toxins such as streptolysin O, Staphylococcus aur.
eus alpha toxin (also known as alpha-hemolysin); addition of other hemolytic factors (eg saponin); or one or more detergents (eg digitonin, Triton X-1).
00, NP-40, n-octyl β-D-glucoside, etc.) at a lower concentration than that used to lyse cells and solubilize the membrane (ie, below the critical micelle concentration). Can be permeabilized. Certain conventional transfection reagents such as DOTAP can also be used. ATP can also be used to permeabilize intact cells, and low concentrations of chemicals commonly used as fixatives (eg formaldehyde) can also be used. All the permeabilizing agents described in this paragraph are, for example, Sigma C
chemical Co. , St. Louis, MO ("Biochemical
s and Reagents for Life Science Rese
Arch. ", Anon, 1999, and the references cited therein for these and other permeabilizing agents).

In certain embodiments of the invention, the subject or biological source may be suspected or have a disease associated with organelle dysfunction, including altered mitochondrial function and mitochondrial dysfunction. Various diseases, and in certain embodiments of the invention it is known that the subject or biological source is neither at risk nor present for such diseases. obtain. Organelle dysfunction is an abnormal, excessive, inefficient, ineffective or deleterious activity at organelle levels (eg biological molecules and macromolecules (eg proteins and polypeptides and their derivatives, Carbohydrates and oligosaccharides and their derivatives, including sugar conjugates (eg glycoproteins and glycolipids)
, Lipids, nucleic acids and cofactors (including ions, mediators, precursors, metabolites, etc.) uptake, release, activity, sequestration, transport, metabolism, catabolism, synthesis, storage or processing defects). Examples of organelle deficiency are lysosomal storage defects (eg mucopolysaccharidosis, I-cell disorders, Wolman's disease and cholesteryl ester storage diseases (eg Du et al., 1998 Mol. Genet. Metab.
64: 126-34)); plasma membrane deficiency (eg, ion channel dysfunction in cystic fibrosis); endoplasmic reticulum storage disease (eg, Kim and Arvan, 1998).
Endocr. Rev. 19: 173-202); diseases associated with Golgi deficiency (eg, ALS, AD, Gonatas et al., 1998 Histochem.
Cell. Biol. 109: 591-600) and other types of organelle dysfunction known to those of skill in the art, but need not be limited thereto.

In certain preferred embodiments, it may be desirable to compare the signal detected according to the method of the invention with a reference signal. The choice of an appropriate reference signal follows criteria familiar to those skilled in the art and can vary depending on the particular cell membrane being assayed and the particular donor-acceptor pair used. For example, the reference signal can be generated by a reference compound such as an ET donor molecule or an ET acceptor molecule, or another reporter molecule that is an indicator as provided herein, and in the absence of sample. Or it can be further made in the presence. Such reporter molecules or indicators can include a detectable compound that can be detected as an indicator, such as one or more of a number of detectable components or the position of the detectable component. For example, by way of example and not limitation, the reference signal can be generated by a reporter molecule that allows normalization of the energy transfer signal detected by the number of cells present (eg, reporter can be cell number). Of any of a number of known indicators, such as selective dyes for cell nuclei, such as propidium iodide or ethidium bromide.

In another particular embodiment, the reference signal is generated by an indicator of mitochondrial mass, mitochondrial number or mitochondrial volume present. For example, if an indicator of mitochondrial mass is selected, a reporter molecule such as nonyl acridine orange, which can also be an ET donor, can be used. Methods for quantifying mitochondrial mass, volume and / or mitochondrial number are known in the art and can include, for example, quantitative staining of a representative biological sample. Quantitative staining of mitochondria typically involves organelle-selective probes or dyes (mitochondrial-selective reagents (eg, fluorescent dyes that bind to molecular components of mitochondria (eg, nonyl acridine orange, MitoTrackers ^™ )) or inner mitochondrial membrane potentials. Potentiometric dyes that accumulate in mitochondria as a function of (eg, Haugland,
1996 Handbook of Fluorescent Probes
and Research Chemicals-Sixth Edition, Molecular
Probes, Eugene, OR)))). As another example, mitochondrial mass, volume and / or number can be determined by morphometric analysis (eg, Cruz-Olive et al., 1990).
Am. J. Physiol. 258: L148; Schwerzman et al., 19
86 J. Cell Biol. 102: 97). These or any other means known in the art for quantifying mitochondrial mass, volume and / or mitochondrial number in a sample are within the contemplated scope of the present invention. For example, the use of such quantitative determinations for the purpose of calculating mitochondrial density is intended, and is not intended to be limiting. In a particularly highly preferred embodiment, the mass of mitochondrial proteins in the sample is determined using well known procedures. For example, one skilled in the art can easily prepare an isolated mitochondrial fraction from a biological sample using established cell fractionation techniques, from which the protein content can be determined as well known in the art. It can be determined using any of a number of protein quantification methodologies.

In other embodiments, the reference signal is a protein abundance (eg, Coomassie blue, fluoresamine, bicinchoninic).
acid)) or according to the abundance of nucleic acids (eg ethidium bromide, acridine orange, methylene blue) and can be generated by a reporter molecule that allows normalization of the detected energy transfer signal. As another example,
The reference signal can be generated by a detectable reporter molecule that is soluble in the liquid medium containing the sample but is unable to cross the cell membrane, and thus acts as a marker (eg, volumetric label) in the extracellular medium. For example, where a highly sensitive instrument (see, eg, supra) can be used to detect the ET signal, such a label may improve quantitative accuracy by calibrating / normalizing the sample volume. Can be enabled. Many compounds suitable for use (eg, reference signals) include
It is known to those skilled in the art. One of ordinary skill in the art can select such compounds as a source of the reference signal in a manner that depends on, among other things, the particular cell membrane potential being assayed and the particular donor-acceptor pair used.

As used herein, detection of the “relative amount” of a signal can include, but is not limited to, detection of the signal for purposes of comparison with a reference signal as provided above. Not limited. Accordingly, detection of the relative amount of signal refers to one of the signals, whether or not any of the other signals detected, such as the following, is a reference signal as provided herein. Detection of only parts (eg 10
Detection of signal with an efficiency of less than 0%) or detection of only part of the signal generated by energy transfer, or of part of the signal relative to the detected signal from another sample (eg a control sample). You can get detection.
Detection of signals according to the methods disclosed herein can include quantification of ET by conventional or arbitrarily assigned units of measurement. In certain embodiments, a signal can be detected over a period of time such that one or more properties of the signal can be analyzed as a function of time. For example, in some embodiments described herein, the signal can be detected over a period of time.
This refers to any method of detecting a sample in a manner that provides more than a single detection event, so that correlation of detection signals at discrete time points can be established. Thus, for example, in certain embodiments, a change in the amount of signal can be detected over two or more time points, and the rate of change in the level of the signal is determined (eg, the signal level is a function of time. , The slope or rate of change of the slope (eg, a linear function) is determined). As in another example, in certain other embodiments, the amount of signal may be cumulatively determined over discrete time intervals to provide a summed signal (eg, an integrated signal). These and other techniques known in the art for analyzing quantitative data, particularly for analyzing such data having transient components, are within the contemplation of the invention. Yes, and described in more detail below.

Accordingly, any of the methods provided by the invention may be modified, eg, standardization of cell number, quantification of cellular proteins or nucleic acids, mass of mitochondria, quantification of mitochondrial proteins or nucleic acids, volume of solution. A reference signal that correlates with a reference parameter of interest for purposes such as a label may also be included. This reference signal, which can be used as an internal standard, need not be the result from energy transfer and can correlate with the desired reference parameters, but can include any signal that does not interfere with the detection of the test / assay signal. In the context of the present invention, a reference compound is one which, if it produces a signal that cannot be quenched from the test / assay signal, or if it is located in the same subcellular compartment as the ET donor compound and the ET acceptor compound, and As such, ET receptor compound or E
When acting as a T-donor compound, it can interfere with the test / assay signal.

An instrument such as the FLIPR ^™ can be set to change between reading signals at two different wavelengths with a circulation time of about 1 second; in this manner a reference signal and a test / assay signal (eg FRET, ΔΨ) can be read over the same time course. However, this reference need not be read at the same time as the test / assay signal. For example, in some aspects of the invention, this requires destroying the cell to detect the reference signal,
And this typically requires that this reference signal be read after the test or assay is complete.

Some non-limiting examples of reference signals include: After testing or assaying, cellular proteins (including mitochondrial proteins) can be measured using methods such as the Bradford assay or the Lowry assay, and the nucleic acid can be propidium iodide (PI), as is known in the art.
) Can be measured through the use of fluorescent dyes such as Nucleic acid can also be measured in living cells. For example, in digitonin permeable cells, when excited by propidium iodine (PI; nucleic acid, peak excitation, 536 nm; peak emission, 617n
m) binds to nucleic acids and cytoplasmic nucleic acids but cannot utilize the mitochondrial matrix and the mitochondrial nucleic acids contained therein; thus PI provides a reference signal for the quantification of cellular nucleic acids. The osmotic compound acridine orange (AO) is used in living cells to discriminate RNA and DNA. This is because acridine orange has different excitation / emission spectra depending on the type of nucleic acid to which it binds (AO: DNA, peak excitation, 500 nm; peak emission, 526 nm; AO: RNA, peak excitation, 460 nm; peak. Luminescence, 650
nm). SYTO stain can also be used to detect nucleic acids in viable cells; the manufacturer of this SYTO stain (Molecular Probes, Inc., Eu).
Gene, OR) show that all of the SYTO stains utilize nuclear and cellular nucleic acids, and some may also utilize mitochondrial nucleic acids; those skilled in the art, such as for example fluorescence microscopy. Utilizing techniques, nucleic acid types can be determined by the use of specific SYTO stains. JC-1 green fluorescence and NAO fluorescence can be used to measure mitochondrial mass in living cells (Mancini et al., Ann. Surg. Oncol. 5: 287-295, 1998; Vayss, respectively.
iere et al., In Vitro Cell. Dev. Biol. 28A: 763
-772, 1992).

The present invention provides diagnostic and prophylactic methods, as well as screening assays (ie, altering a monitored process or condition (eg, mitochondrial membrane potential) (ie, increasing in a statistically sufficient manner, or A method of identifying a drug that is reduced). Diagnostic applications include methods of assaying cellular processes or conditions (eg, cell membrane potential (eg, mitochondrial membrane potential)). Here, a biological sample containing a cell membrane or subcellular compartment (eg, organelle (eg, mitochondria)) has a disease or disorder, or is prone to, or suspected of having, a disease or disorder (eg, ,
Having an increased risk of, or the likelihood of developing, a risk-related disease in a reference population), wherein further the process or condition is
It can be altered in relation to the process or condition determined in a control sample from a patient known not to have the disease or disorder. For diagnostic purposes,
Methods in which a biological sample having a cell membrane or subcellular compartment is taken from a patient known to have a disease or disorder that alters the intracellular process or condition being monitored. In such prophylactic applications, for example, biological samples from patients are prepared and affect some or not all, but not all, monitored intracellular processes or conditions. Are tested for their response to agents known to be. The desired response of the biological sample to a particular drug indicates that the patient from whom this sample is taken has the best response to the treatment associated with the positive response corresponding to that treatment. In a related aspect, pharmacogenetics studies using the present invention are used to determine the correlation between different treatments and the particular measurements produced by the present invention.

Non-limiting examples of diseases or disorders that are believed to involve altered function or dysfunction of subcellular compartments include Alzheimer's disease, Parkinson's disease, type II diabetes and lysosomal storage disorders. When the subcellular compartment of interest is mitochondria, the preferred biological sample is a cytoplasmic hybrid (eg, plasmacytoplasmic hybrid cells contain a common nuclear component but different individuals (ie, patient and control)). With the mitochondria of origin). Methods for preparing and using cytoplasmic hybrids are described in US Pat. No. 5,888,438, published PCT application WO
95/26973 and WO 98/17826, King and Attard.
i (Science 246: 500-503, 1989), Chomyn et al.
Mol. Cell. Biol. 11: 2236-2244, 1991), Mil.
ler et al. (J. Neurochem. 67: 1897-1907, 1996),
Swedlow et al. (Annals of Neurology 40: 663.
-671, 1996), Cassarino et al. (Biochim. Biophy.
s. Acta 1362: 77-86, 1997), Swedlow et al. (Ne).
urology 49: 918-925, 1997), Sheehan et al. (J.
Neurochem. 68: 1221-1233, 1997), and She.
ehan et al. (J. Neurosci. 17: 4612-4622, 1997) (
All of which are incorporated herein by reference).

The term “screening” refers to the use of the invention to identify agents that affect intracellular processes or conditions that are monitored in a negative or positive manner. The cell or organelle is treated with an agent thought to affect the intracellular process or condition being monitored, and the response of the subcellular compartment of interest to this agent is monitored and the vehicle used to transport the agent. Compared to control sample treated alone. Agents that affect the intracellular process or condition being monitored result in an altered response of the subcellular compartment of interest as compared to the response in the control sample. In certain aspects of the invention, agents that act in a species-specific manner are identified by the screening methods of the invention.

The present invention relates to energy transfer between a chemically distinct and independent ET donor molecule and an ET acceptor molecule, which comprises (i) both ET and ET acceptor molecules. Localize in the same subcellular compartment; (ii) one ET molecule (ie, ET donor or ET acceptor) localizes in a particular subcellular compartment and another ET
If the molecule (ie the ET acceptor or ET donor) is localized in the membrane forming one border of the subcellular compartments; or (iii) one ET molecule (ie the ET donor or ET acceptor) Other ET molecules (ie,
This can occur when the ET acceptor or ET donor) binds transiently or otherwise to the subcellular compartment.

In state (i), a change in the efficiency and / or rate of energy transfer between the ET donor molecule and the ET acceptor molecule is dependent on the state or occurrence of a given process within the subcellular compartment of interest. Correlate with change in change. Non-limiting examples of aspects of the invention described in more detail below assay mitochondrial membrane potential (ΔΨ) or pH potential (ΔpH), photosynthesis within chloroplasts, and the formation of secondary lysosomes. There is a method. According to the invention, if at least one subcellular compartment of a particular cell type accumulates and / or retains ET donor or ET acceptor molecules to a greater extent than occurs in other cell types, then according to the invention Any method can be used to detect the presence of a particular cell type in a biological sample.

In state (ii), a change in the rate of energy transfer between the ET donor molecule and the ET acceptor molecule affects the cell membrane containing either the ET donor molecule or the ET acceptor molecule. (Eg, altering cell membrane potential), and / or correlating with processes affecting subcellular compartments bound by the cell membrane, which compartment is another member of the ET molecule pair (eg, ET acceptor molecule or ET donor). Body molecule). Non-limiting examples of this aspect of the invention described in more detail herein include: mitochondrial pore translocation.
methods of monitoring transition (MPT) and virus non-coating processes.

In the context of (iii), the change in the rate of energy transfer between the ET donor molecule and the ET acceptor molecule is due to a detectably labeled molecule (eg, either the ET donor or the ET acceptor). Labeled with) or labeled subcellular compartment (
(Eg, labeled with either an ET acceptor or an ET donor), or associated with a separate form therefrom. As a non-limiting example of such an embodiment of the invention described in more detail below, a method for monitoring the binding of Bcl-2 protein to the outer mitochondrial membrane, or the separation of cytochrome c therefrom. Is mentioned.

Donor-Acceptor Pairs According to the present invention, paired ET molecules are provided. Where each pair is ET
It includes a donor molecule and an ET acceptor molecule. As described herein, the combination of energy donor compound (ET donor molecule) and energy acceptor compound (ET acceptor molecule) that is available for the ET-based assay of the present invention is determined. There are several criteria for: Additional criteria design this assay to determine the specific intracellular state or activity (eg, mitochondrial inner membrane potential (ΔΨ or ΔΨm), binding of a specific intracellular molecule or factor to a specific organelle,
It may be applied specifically when monitoring the release of a particular intracellular molecule or factor, such as from organelles.

One criterion for determining suitable ET donor-acceptor pairs for use in accordance with the present invention is that the energy emission spectrum of the ET donor molecule is at least one of the energy absorption spectra of the ET acceptor molecule. Should overlap, so that energy transfer from the donor to the acceptor may occur. Typically, the ET donor compound is the excitation peak wavelength of the acceptor compound (herein, “λA (ex)”).
), The wavelength of the emission peak (“λD (em)” in this specification) is within several nm. That is, the difference between D (em) and A (ex) is typically about 70n.
m to less than about 20 nm and typical values are 60 nm or less, 50 nm or less, 40
nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 n
This difference Δ = λD (em) −λA (m or less, 5 nm or less, or 1 nm or less
ex). However, if the excitation or emission is plotted as a function of wavelength, then certain compounds suitable for use as ET donor or ET acceptor molecules will have D () greater than those described herein. em) and A (e
x) may have a broad peak so that it can be detectably transferred between a particular paired ET donor molecule and ET acceptor molecule. For example, a particular donor-acceptor pair may have very low energy transfer between them, as long as the ET donor and ET acceptor are sufficiently close to each other to cause a detectable energy transfer. Inefficiency (ie, when one or both of the ET donor and ET acceptor are used with light having wavelengths far from the excitation peak wavelength and / or the emission peak wavelength for the ET molecule) However, it may be suitable for the ET methodologies provided herein. In the presence of fluorescence resonance energy transfer, one of ordinary skill in the art will perform undue experimentation so that the selection of the appropriate ET donor-acceptor pair can be accomplished in accordance with established criteria and the teachings provided herein. Without it can be easily determined.

For example, conventional screening may involve at least one as disclosed herein for the purpose of determining whether a detectable FRET signal may be generated.
It can be used by combining one candidate ET donor molecule and a candidate ET acceptor molecule in solution (eg, in the absence of a biological sample). One of the donor and acceptor in the subcellular compartment for a particular donor-acceptor combination
Selective accumulation of one or both may depend on binding of the donor and / or acceptor to molecules present in the subcellular compartment, and other donors in such compartment-
The accumulation of receptor pairs cannot be involved in such binding. Thus, screening for specific donor-acceptor pairs for their enhancement of detectable FRET signal in solution is a matter of donor and / or acceptor in the biological sample intended for use in the methods of the invention. Of at least one suitable biomolecule (eg, protein-containing or peptide-containing species, lipid-containing species, selected by one of skill in the art based on familiarity with the nature of and / or properties of the subcellular compartments,
Nucleic acid-containing species or sugar-containing species) is added to the solution. Without wishing to be bound by theory, the concentration of ET donor molecule and ET acceptor molecule used in such pilot experiments to detect the FRET signal is, in certain such examples, The concentrations used in the methods of the invention provided herein may be exceeded. However, even when substantially lower concentrations of ET molecules are first contacted with the sample, similarly detectable concentrations of such ET molecules will be present in the subcellular compartments of the samples described herein. Can accumulate. One of skill in the art will also appreciate that the fluorescence spectral properties of the ET donor and ET acceptor molecules as a function of the solution and sample conditions used (eg, solvent, solvent and ionic strength selected, pH, sample properties, etc.). Easily understand that it can change.

Another criterion that is useful in selecting an appropriate ET donor-acceptor pair for use in accordance with the present invention is that the emission signal from the excited ET acceptor compound is the excited ET donor compound. It must be able to be distinguished from the luminescent signal from The luminescence signal from the excited donor is, for example, (1) when the wavelength of the luminescence signal from the excited acceptor is sufficiently distinguished from the wavelength of the luminescence signal from the excited donor, or (2) the acceptor Can be so distinguished if they quench the emission signal from an excited donor.

Various classes of compounds may act as ET acceptor molecules and ET donor molecules according to the present invention, and the acceptors and donors may, but need not, belong to the same class of compounds. . For example, a fluorescent protein may be an ET donor molecule for an ET acceptor that is a small organic compound, or ET for an acceptor that is a different fluorescent protein, as long as it meets the other criteria needed for this assay. It can act as a donor molecule. Table 1 lists abbreviations for ET donor compounds and ET acceptor compounds among others, and Table 2 lists some ET donor-acceptor pairings suitable for ET-based assays (various greens). Most of the compounds listed in Table 2, except the fluorescent protein derivative, are found in the Molecular Probe.
s, Inc. , Eugene, OR).

[0070]

[Table 2] * Minor Excitation Peak or Minor Emission Peak ** JC-1 Monomer vs. JC-1 Aggregate: At Higher Concentration (Aqueous Solution> 0.1 μM)
Or in mitochondria with higher potential and with J-aggregate: parent compound, it has different spectral properties.

A variety of small hydrophilic molecules can serve as ET donor and ET acceptor molecules. Such compounds may be used when the donor and / or acceptor compounds are desired to undergo energy transfer at water-based subcellular sites or compartments. In some aspects of the invention, it may also be desirable to predominantly accumulate such compounds at water-based subcellular sites or compartments. Some such compounds are known to accumulate predominantly at specific subcellular locations.
Additionally or alternatively, a moiety that directs the compound to a subcellular location can be attached to a donor or acceptor moiety to produce a donor or acceptor compound that can predominantly accumulate at the selected subcellular location. For example, published PCT application WO98 / 1
7826, incorporated herein by reference, describes methods for attaching mitochondrial-directing moieties to various compounds.

Small lipophilic molecules may be used when it is desired to cause the donor and / or acceptor compounds to predominantly accumulate in the cell membrane, such membranes typically being a significant portion of the lipid. It consists of double layers. Additionally or alternatively, the lipid or lipophilic molecule is
It may bind to a donor or acceptor moiety to produce a donor or acceptor compound that can accumulate predominantly in the cell membrane.

Examples of proteins that can serve as donor and acceptor compounds include “FLASH
A fusion protein (Grif containing a fluorescein arsenic helical binder) sequence
fin et al., Science 281: 269-272, 1998), or aequorin protein or green fluorescent protein (Kendall et al., Trend).
in Biotechnology 16: 216-224, 1998, and the references cited therein). As used herein, "green fluorescent protein" refers to wild-type green fluorescent protein (wild-type GFP), as well as blue-shifted derivatives, blue-green-shifted derivatives, red-shifted derivatives of wild-type GFP. And yellow-shifted derivatives (designated BFP, CFP, RFP and YFP, respectively; see published PCT application WO98 / 06737). Table 2 shows
Amino acid changes in various green fluorescent protein derivatives, and their GF
It includes a description of the excitation and emission maximum wavelengths of each of the P-derivatives.

To produce an expression construct producing an aequorin, GFP or FLASH fusion protein that accumulates at the organelle or other subcellular site of interest, an expression vector containing a nucleotide sequence suitable for gene expression is described below (1 And (2): (1) a first nucleic acid encoding a GFP derivative or a FLASH polypeptide, and (2) a peptide sequence that directs the protein to the organelle or other subcellular site of interest. A second nucleic acid that encodes (ie, a "targeting sequence"). Here, the first and second nucleic acids are linked such that they have a common reading frame that includes both nucleic acids. Such fusion proteins may be based on the characteristics of the particular targeting sequence used for a given purpose, such as in the inner membrane of a particular membrane within the cell (for example, the nuclear membrane, or the organelles of mitochondria and chloroplasts or Outer membrane), or other specific subcellular location. Table 3 lists some non-limiting examples of intracellular sites where the donor and acceptor compounds listed in Table 2 accumulate.

[0075]

[Table 3] A further criterion is that the donor and acceptor compounds must accumulate in subcellular compartments at the same site, which can cause ET, or at an acceptably adjacent site. By "acceptably adjacent" is meant that such sites are sufficiently close in proximity for ET to occur. Such sites are separated from each other by about 100 Å (Å) to about 10 Å or less, depending on the donor-acceptor pair of the compound, and are typically about 80Å, 60Å, 50Å, 40Å, 30Å, 25
Å, 20 Å, 15 Å, 10 Å, 5 Å or less, preferably 70 Å or less, more preferably 50 Å or less, and most preferably 40 Å
Below are separated from each other. In each case, the relationship between (i) the distance between one ET donor molecule and one ET donor molecule to (ii) the ability of ET to occur is well established (see, for example, Haugland, 1996. Handb
look of Fluorescent Probes and Rear
ch Chemicals, 6th Edition, Molecular Probes, Eu
Gene, OR), one skilled in the art will readily understand that the donor-acceptor intermolecular distance is the major determinant of the efficiency of ET.

As a non-limiting example, one subcellular site of interest is the organelle known as mitochondria. Mitochondria are outer membranes that are exposed to the cytoplasm and are capable of transiently or stably associating with various cytoplasmic factors, arranged as shown in FIG.
It includes the intima, the transmembrane space between the intima and adventitia, and the matrix (the compartment within the intima). For mitochondria, at admissibly adjacent sites, (i) an outer membrane and a cytoplasm containing cytoplasmic factors associated with the outer membrane; (ii) an outer membrane and an intermembrane space; (iii) an intermembrane space and an inner membrane. And (iv) intima and matrix (
Including the factors within the matrix).

For example, but not limited to, mitochondria, a GFP fusion protein derivative is a cytochrome c oxidase subunit IV protein sequence (Llopis).
Et al., Proc. Natl. Acad. Sci. U. S. A. 95: 6803-6
808, 1993) to the mitochondrial matrix using cytochrome c protein sequences (Mahajan et al., Nature Biotech. 16:54).
7-552, 1998) and into the intermembrane space and hexokinase (Sui et al., Arch. Biochem. Biophys. 345: 111-125, 19).
97), Bcl-2 or Bax (Mahajan et al., Nature Biot.
ech. 16: 547-552, 1998) protein sequence is used to target the outer membrane of mitochondria. The GFP fusion protein also has 3
-Oxoacyl-CoA-thiolase (Zhang et al., Biochem. Bio.
phys. Res. Commun. 242: 390-395, 1998), OS
CP (Prescott et al., FEBS Letts. 411: 97-101, 1
997), and BNIP3 (Yasuda et al., J. Biol. Chem. 27).
3: 12415-12421, 1998) protein sequence has been used to target mitochondria. Aequorin fusion protein derivatives have been targeted to mitochondria using the cytochrome c oxidase protein sequence (Pinton et al., Biofactors 8: 243-25.
3, 1998; Rizzuto et al., Nature 358: 325-327,1.
992). Other fusion proteins include protein sequences derived from mitochondrial (or bacterial) thiolase (Arakawa et al., J. Biochem., Tokyo).
, 107: 160-164, 1990), F0-ATPase subunit 9 (
J. Biol. Chem. 271: 25208-25212, 1996), manganese superoxide dismutase (Balzan et al., Proc. Natl. A).
cad. Sci. U. S. A. 92: 4219-4223, 1995), and p-450 (SCC) (Kumamoto et al., J. Biochem., Tokyo).
o, 105: 72-78, 1989) have been described.

For example, but not limited to, in chloroplasts, the fusion protein may be a SCE70 heat shock protein targeting sequence (Wu et al., J. Biol. Chem. 26).
8: 19384-19391, 1993). For example, a sequence derived from Rieske iron-sulfur protein (Maduen
o et al. Biol. Chem. 269: 17458-17463,1994)
Other targeting sequences, such as, direct the fusion protein through the thylakoid membrane.

If dual targeting to mitochondria and chloroplasts is desired, several fusion proteins containing dual targeting sequences have been described (C
Reissen et al., Plant J. et al. 8: 167-175, 1995; Huan.
g., et al., Plant Cell 2: 1249-1260, 1990). Conversely, if plant cells are used and it is desired to target only to mitochondria or chloroplasts, care must be taken to ensure that double targeting sequences are not used.

For example, but not limited to, in the case of the nucleus, the aequorin fusion protein derivative has been targeted to the nucleus using a nucleoplasmic protein sequence (Badminto).
n et al. Biol. Chem. 271: 31210-31214, 1997).
.

For example, but not limited to, the endoplasmic reticulum (ER), an aequorin fusion protein derivative has been targeted to the endoplasmic reticulum using a calreticulin protein sequence (Kendall et al., Biochem. Biophys. Res.
Commun. 189: 1008-1016, 1992).

For example, but not limited to, in the Golgi apparatus, aequorin fusion protein derivatives include galactosyltransferase, SNAP-25, connexin and 5-
It has been targeted to the Golgi plasma membrane using the HT _1A receptor protein sequence (Burton et al., Mol. Cell. Biol. 7: 419-43.
4, 1996; Marsault et al., EMBO J .; 16: 1575-1581
Daguzan et al., Int. J. Dev. Biol. 39: 653
-657, 1995). The GFP fusion protein is targeted to the Golgi apparatus using the galactosyltransferase protein sequence (L
lopis et al., Proc. Natl. Acad. Sci. U. S. A. 95: 6
803-6808, 1993).

For whole cell assays, another criterion is the accumulation of ET donor and acceptor molecules,
Preferably, it must occur predominantly at sites within the mitochondria or at sites of interest in either the organelle or subcellular compartments. But,
In particular, if the donor and acceptor do not accumulate at the same secondary intracellular site (ie, so that ET cannot occur at that secondary site), or background ET
If the amount of inducing signal is small enough that the organelle-specific phenomenon of interest can be understood despite accumulation of the compound at the secondary site, some of these compounds at other secondary sites within the cell Accumulation of is acceptable. Furthermore, most, if not all, of the assays described herein can be adapted for use with isolated organelles, where predominant accumulation is not the norm.

Instrumentation for Detecting Energy Transfer Various devices can be used in the methods of the invention to excite the donor compound and measure the emission from the acceptor compound. Which device is applicable to a particular donor-acceptor pair depends on factors such as: (1) the wavelength at which the donor compound is excited, preferably at or near λD (ex). Energy to the sample; (2) the energy in the emission spectrum of the acceptor compound needs to be measured, preferably at or near λA (em); (3) assayed with a given program Sample type; and (4) number of samples assayed in a given program.

With respect to factors (1) and (2), the spectrum of energy applied to the sample to excite the donor compound and the spectrum of energy emitted and measured by the excited acceptor compound in the sample Generally, it is decided which type of device will be used. For example, λD (em) is not the same as λA (em), but the minimum acceptable amount of difference between these two values is influenced by the instrumentation used, among other factors. That is, λD (em) is equal to λA (e
m), a device capable of resolving close wavelengths is needed, and λD
Assays using donor-acceptor pairs where the difference between (me) and λA (em) is less than about 3 to less than about 5 nm requires high resolution equipment. Conversely, λD (me) and λ
Assays using donor-acceptor pairs with a difference between A (em) greater than about 50 to about 75 nm require medium to low resolution instruments.

In particular, with respect to factor (2), the type of energy emitted by the excited acceptor compound and measured in the sample generally determines which type of instrument is used. By definition, a fluorometer is a device that measures fluorescence energy and should therefore be part of the instrumentation. A fluorometer is a relatively simple, manually operated instrument that contains only a few sample tubes at a time, and allows for somewhat more complex, larger numbers of samples, such as 96-well microplates (
For example, fmax ^™ Fluorescence Plate Reader, Molecular Devi
ces Corp. , Sunnyvale, CA; or Cytofluor fluorometric plate reader, model # 2350, Millipore Corp .;
, Bedford, MA), manually operated or robotic instruments, or complex robotic instruments (eg, FLIPR) containing multiple samples of various formats such as 96-well microplates. ^TM device; see below)).

Regarding the sample type assayed in a given program, which is factor (3), different formats are suitable for different types of samples. For example, 96-well microplates are suitable where cells of interest or isolated organelles adhere to the material of the microplate or any material applied to the wells of the microplate; however, fluorescence of plastics Is about 400n
Larger background components occur at excitation wavelengths below m. For measurements involving non-adherent cells or organelles, or soluble extracts prepared therefrom, instruments capable of reading the fluorescent signal in glass or polymer tubes or tubing are preferred. Regardless of which type of format is used, it should allow the introduction of donor and acceptor compounds, and control and evaluated compounds into the sample at the appropriate time points. .

Factor (4), the number of samples assayed in a given program, affects how the instrument is automated. For example, robotic or semi-robot instruments are preferred when high throughput (HTS) assays of large numbers of samples are required. However, a significant number of samples may be processed manually, especially when formats that accommodate large sample numbers (eg, 96-well microplates) are used.

Depending on the assay, a fluorometric imaging plate reader (FLIPR ^™ ) instrument (Molecular Devices, Sunnyvale, CA) is often the instrument of choice for the ET-based assays of the invention. FLIP
R ^™ system (http: //www.moleculardevices.c)
om / pages / flipr. html) has the following desired characteristics: The system is a water-cooled argon ion laser irradiation and a cooled CC.
The combination with a D-camera is used as an integrated detector which accumulates the signal over the period in which the signal is exposed to the image and, as a result, its signal-to-noise characteristic causes Generally superior to the signal-to-noise characteristics of optics; this system also uses proprietary cell-layer isolation optics, which allows for discrimination of signals on cell monolayers, and therefore undesired cells Reduce background fluorescence outside; this system provides data in real time and also kinetic data (ie, readings at multiple time points); this system provides 96 wells The ability to simultaneously stimulate and read all 96 wells of a microplate; Providing precise control of temperature and humidity; the system is equipped with an integrated, state-of-the-art 96-well pipettor, which has precise volume to eliminate carryover between experiments. Using a dispensable tip that can be used to aspirate, dispense, and mix fluids from a microplate; and in the case of the FLIPR ³⁸⁴ instrument, the system is
The sample assay may be adapted to be performed in a robotic or semi-robot manner, which allows for the shortest number of samples (eg, about 10 per day).
Providing analysis on up to 0 96-well microplates).

Monitoring Conditions or Processes Within Subcellular Compartments The term “subcellular compartment” refers to any intracellular space that is maintained in at least partially separated conditions for at least some of its time. Some types of physical barriers, typically lipid bilayers, form a boundary between a given subcellular compartment and other cellular compartments. The perimeter of a subcellular compartment may be permeable, impermeable or semipermeable to molecules inside or outside this subcellular compartment. Subcellular compartments include, but are not limited to, known organelles (eg, in eukaryotic cells, nuclei, nucleoli, mitochondria, chloroplasts, endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus, etc.). Not done. The present invention also finds use in extracellular subcellular structures that interact with cells and / or are internalized by cells, including but not limited to viruses and other intracellular parasites. Can be done. Some of the subcellular compartments that may be monitored or assayed using the present invention, and in particular the application for each such subcellular compartment, are described in further detail in the following subsections.

Mitochondria One subcellular compartment of particular interest is the organelle known as mitochondria. Mitochondria are the major energy source in cells of higher organisms and provide direct and indirect biochemical regulation of respiratory, oxidative, and metabolic processes in a wide array of cells. These are electron transfer chains (
ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and also of central mitochondrial role in intracellular calcium homeostasis. Be the basis.

In addition to their role in energy production in proliferating cells, mitochondria (or at least mitochondrial components) are involved in programmed cell death (PCD), also known as apoptosis (Newmeyer).
Et al., 1994, Cell 79: 353-364; Liu et al., 1996, Cel.
86: 147-157). Apoptosis is clearly required for normal nervous system development and immune system function. Furthermore, some disease states are thought to be associated with either insufficient or excessive levels of apoptosis (eg cancer and autoimmune diseases in the first example, and in the latter case). Neurodegeneration in stroke injury and Alzheimer's disease). For a general review of apoptosis and the role of mitochondria in apoptosis, see Green and Reed (1998, Science 281: 1).
309-1312), Green (1998, Cell 94: 695-598).
) And Kromer (1997, Nature Medicine 3: 6).
14-620). Therefore, factors that influence apoptotic events, including factors associated with mitochondrial components, have various therapeutic, therapeutic, palliative, rehabilitative, preventative (preventative) uses.
ve), may have prophylactic, or disease-disturbing uses.

Various apoptogens are known to those of skill in the art (see, eg, Green et al., 1998 Science 281: 1309 and references cited therein), and examples include: Can be
Without limitation: tumor necrosis factor-α (TNF-α); Fas ligand; glutamate; N-methyl-D-aspartate (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al. 1997 J. Ce
ll. Biol. 138: 449-469); Paraquat (Cosanti)
ni et al., 1995 Toxicology 99: 1-2); ethylene glycol; protein kinase inhibitors (eg, staurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrine chloride, genistein).
1- (5-isoquinolinesulfonyl) -2-methylpiperazine; N- [2- (
(P-Bromocinnamyl) amino) ethyl] -5-5-isoquinolinesulfonamide; KN-93; quercitin; d-erythro-sphingosine derivative; UV irradiation; ionophore (eg, ionomycin and valinomycin); MAP kinase inducer (Eg, anisomycin, anandamine); cell cycle blocker (eg, aphidicolin, colcemid, 5-fluorouracil, homoharringtonine); acetylcholinesterase inhibitor (eg, berberine); anti-estrogen (eg, tamoxifen); Oxidation promoters (eg tert-butyl peroxide, hydrogen peroxide); free radicals (eg nitric oxide); inorganic metal ions (eg cadmium); D A synthesis inhibitors (e.g., actinomycin D); D
NA intercalators (eg, doxorubicin, bleomycin sulfate, hydroxyurea, methotrexate, mitomycin C, camptothecin, daunorubicin); protein synthesis inhibitors (eg, cycloheximide, puromycin, rapamycin); agents that affect microtubule formation or stability (Eg, vinblastine, vincristine, colchicine, 4-hydroxyphenylretinamide, paclitaxel); Bad protein, Bid protein and Ba.
x protein (eg Jurgenmeier et al. 1998 Proc. Na
t. Acad. Sci. USA 95: 4997-5002 and references cited therein); calcium phosphate and inorganic phosphate (Kr.
Oemer et al., 1998 Ann. Rev. Physiol. 60: 619).

Ultrastructural characterization of mitochondria appears to form mitochondrial outer membrane, an attachment to the outer mitochondrial membrane at multiple sites, which acts as an interface between organelles and the cytosol, a highly folded The presence of the inner mitochondrial membrane and the transmembrane space between these two mitochondrial membranes is revealed (see Figure 2). The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix (for a review see, eg, Ernster et al., 1981.
J. Cell Biol. 91: 227s). Initially hypothesized to exist as an unfolded inner mitochondrial membrane, cristhe
Recently, using three-dimensional electron tomography, it has been characterized that it also includes a tubular conduit, which can form a network and is open circular (30 nm diameter).
) Can be attached to the intima by a conjugation (Perkins et al., 1997, Jour.
nal of Structural Biology 119: 260). The outer membrane is freely permeable to ionic and nonionic solutes with molecular weights less than about 10 kilodaltons, whereas the inner mitochondrial membrane is selective for many small molecules, including certain cations. It exhibits targeted and regulated permeability and is impermeable to large molecules (greater than about 10 kDa).

Of the five multi-subunit protein complexes, four that mediate ETC activity (complexes I, III, IV and V) localize to the inner mitochondrial membrane. The remaining ETC complex (Complex II) is located in the matrix. In at least three different chemical reactions known to occur within the ETC, protons migrate from the mitochondrial matrix across the inner membrane into the intermembrane space. This imbalance of charged species results in an electrochemical potential of about 220 mV called the "proton driving force" (PMF), which is often represented by the notation ΔΨ or ΔΨm. ΔΨm represents the sum of the potential and pH potential (ie, pH difference) across the inner mitochondrial membrane (eg, Ernster et al., 1981 J. Ce.
ll Biol. 91: 227s and references cited therein).

ΔΨm provides the energy to phosphorylate adenosine diphosphate (ADP) by ETC complex V to yield ATP, a process that stoichiometrically couples proton transport into the matrix. . ΔΨm is also a driving force for influxing cytosolic Ca ²⁺ into mitochondria. Under normal metabolic conditions, the inner membrane is impermeable to proton transfer from the intermembrane space into the matrix,
The TC complex V remains as the only means by which protons can be returned to the matrix. However, if the integrity of the inner mitochondrial membrane is compromised (as occurs during mitochondrial permeability change (MPT) with certain diseases associated with altered mitochondrial function), the protons may produce ATP. The conduit of complex V may be bypassed, thereby uncoupling the breath from energy production. During MPT, ΔΨm plummets, and the mitochondrial membrane has the ability to maintain an equilibrium distribution of one or more ionic species or other solutes (ie, small solutes (eg, ionic Ca ²⁺ , Na ⁺ , K ⁺ , H ⁺ ) and / or the ability to selectively regulate permeability to large solutes (eg proteins).

Loss of mitochondrial membrane electrochemical potential may be the result of mechanisms such as free radical oxidation, or may be due to direct or indirect effects of mitochondrial gene products and / or extramitochondrial gene products. . Loss of mitochondrial potential appears to be a key event in disease progression associated with altered mitochondrial function. The diseases include: degenerative diseases such as Alzheimer's disease; diabetes mellitus; Parkinson's disease; Huntington's disease; ataxia; Leber's hereditary optic atrophy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and seizures. (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonus epilepsy red rag fiber syndrome. In order to provide improved therapies for such diseases, agents that limit or prevent loss of mitochondrial membrane potential (ΔΨ _m ) are
Can be advantageous. The present invention provides a new approach to identify useful agents for such diseases. The present invention meets the need for an assay that allows rapid screening for agents that can alter mitochondrial membrane potential,
And provides other related benefits.

Assays for Measuring Changes in Subcellular Compartment Parameters ET-based assays have shown that changes in state or some activity in the subcellular compartment or site are reduced or increased (eg, mitochondrial ΔΨ or cells). Additional criteria for donor-acceptor compounds when designed to measure the presence or absence of factors that either transiently bind to internal positions or are released from intracellular sites are: One (either the donor compound or the acceptor compound) must accumulate in and / or the subcellular compartment or site in a manner that depends on the selected parameter or activity. It must be released from other compounds in the subcellular compartment (receptors The presence of other donor) must be independent of the selected parameter or activity.

Compounds whose mitochondrial concentration depends on ΔΨ are for illustration and not limitation, TMRM (Farkas et al., Biophys.
J. 56: 1053-1069, 1989), TMRE (Ehrenberg et al., Biophys. J. 53: 785-794, 1988), Rhodamine 123.
(Scaduto et al., Biophys. J. 76: 469-477, 1999).
, Ethidium bromide (Coppey-Moisan et al., Biophys. J. 71.
: 2319-2328, 1996), DASPMI (4-Di-ASP and 2).
-Di-1-ASP) and DASPEI (Rafael et al., FEBS Let).
t. 170: 181-185, 1984). Compounds whose mitochondrial concentration does not depend on ΔΨ are for illustration and not limitation, NAO (Mafta et al., Biophys. Res. Commun.
164: 185-190, 1989), MitoTracker® G.
reen FM and MitoFluor ^™ Green (Haugland, H
andbook of Fluorescent Probes and Re
search Chemicals, 6th Edition, Molecular Probes
s, Inc. , Eugene, OR, 1996, p. 269), and DAPI (
Copy-Moisan et al., Biophys. J. 71: 2319-2328
, 1996). Both the plunge and consumption of ΔΨ can be monitored using such compounds. As used herein, “ΔΨ plunge” refers to the rapid disappearance of ΔΨ, that is, ΔΨ is an agent that causes mitochondria to induce a sudden drop in mitochondrial membrane potential (eg, CCCP or FCCP or , Any other drug that can quickly bring ΔΨ _m to 0), and then to 0 within minutes.
To reach. The term “ΔΨ consumption” refers to agents whose mitochondria induce consumption of mitochondrial membrane potential (eg, ionomycin, thapsigargin,
Atractyloside, A23187, 4-bromo-A23187, adenine nucleotide translocator inhibitor, mitochondrial electron transfer chain (ETC)
Inhibitors of complex I, inhibitors of ETC complex II in the presence of complex I substrate, partial inhibitors of other ETCs, or mitochondrial calcium concentrations as a result of elevated intracellular cytosolic free calcium concentrations Does not produce a ΔΨ reaching 0 within minutes after treatment with other drugs (but this may occur over a longer time frame or after repeated exposure). , A slow decrease of ΔΨ. The person skilled in the art is aware of any number of mitochondrial ETC inhibitors characterized as to which ETC component can be compromised. For further disclosures relating to measurements of mitochondrial membrane potential, agents that induce a sudden drop in mitochondrial membrane potential, and agents that induce consumption of mitochondrial membrane potential, see US Application Serial Nos. 09 / 161,172 and 0.
See 9 / 185,904.

Using mitochondria as an example, various factors are either (1) transiently associated with the outer membrane of mitochondria, or (2) typically located at sites within the mitochondria, such as: Mitochondrial pore migration (mitochondrial)
pore transition (MPT) or apoptosis (also known as programmed cell death, PCD; for a review, Green et al., Science).
281: 1309-1312, 1998) and is either released from mitochondria during events. Class (1)
Examples of proteins belonging to the category Hexokinase II, as well as Bcl-2, Bc
l-X _L, include other members of the Bax and bcl-2 gene family (Kroemer, Nature Med.3: 614-620,1997; N
arita et al., Proc. Natl. Acad. Sci. U. S. A. 95: 1
4681-14686, 1998). Examples of class (2) factors released during MPT or apoptosis include cytochrome c (Yang et al., Science).
275: 1129-1132, 1997; Kluck et al., Science 27.
5: 1132-1136, 1997), procaspase-2 and -9 (Sus.
in et al. Exp. Med. 189: 381-394, 1998), as well as apoptosis-inducing factor (AIF; Susin et al., J. Exp. Med. 184:
1331-1341, 1996; Susin et al., J. Am. Exp. Med. 186:
25-37, 1997). Nucleic acids containing nucleotide sequences encoding these proteins have been used to construct fluorescent derivatives that exhibit the same transient binding to or release from mitochondria as the corresponding parental proteins, FLASH, aequorin. , Or a fusion protein with a green fluorescent protein (eg, wild type GFP, BFP, CFP, REP and YFP). For example, a hexokinase II fusion protein that binds to the outer mitochondrial membrane (Sui et al., Arch. Biochem. Biophys. 345: 11.
1-125, 1997), and GFP (Mahajan et al., Nature B.
iotech. 16: 547-552, 1998) or other proteins (Ny
e et al., Mol. Cell. Biol. 10: 5763-5771, 1990) have been described that localize cytochrome c fusion proteins to the intermembrane space of mitochondria. FLASH, aequorin, and green fluorescent protein fusion proteins are designed to monitor the extent and / or rate of mitochondrial binding or mitochondrial release of factors with different biological functions.
Used as a donor or acceptor compound in ET-based assays.

The ET-based methods of the invention have certain advantages over other methods for assaying ΔΨ _m . For example, a method utilizing a single potential fluorophores (i.e., fluorophore that accumulate in mitochondria [Delta] [Psi] _m dependent manner) is, [Delta] [Psi] _m
It may be necessary for the fluorophore to be present at a concentration that is toxic when an agent affecting A. is introduced (see, eg, US Pat. No. 5,169,788). In contrast, the ET-based assay of ΔΨ _m of the present invention can be carried out using lower, non-toxic doses of fluorophore. Moreover, plasma membrane potential contributes to the signal in assays where a single potentiometric fluorophore is used, whereas the ET-based assay of the present invention is specific for changes in mitochondrial membrane potential.

[0102]   The fluorescence emission detected is typically compared to a reference signal. ΔΨ _m For the quantitative measurement of_mAnd smell of mitochondria
Can be the signal observed, and one or more such reference signals
Can be done. Alternatively, ΔΨ_mUnder certain specific conditions ΔΨ_mTo evaluate the changes in
The same type of mitochondria (eg, from the same subject or biological source)
ΔΨ in the mitochondria of_mOr different types of mitochondria (
Δ in, for example, mitochondria from separate subjects or biological sources)
Ψ_mCan be evaluated in comparison with. Specific embodiments of the invention are described in more detail below.
As mentioned, different reference signals may be used.

Chloroplast Chloroplast is an organelle found in plant cells where photosynthesis occurs. In addition to being an integral part of the metabolism of plant cells, photosynthesis is an important process that affects many other organisms alike. The reason for this is twofold: photosynthesis converts atmospheric CO ₂ into bioavailable carbohydrates (CHO).
) “Fix” on _n molecules and also produce O ₂ required by all aerobic organisms.

Like mitochondria, chloroplasts have bilayer membranes (outer and inner membranes), contain their own DNA, and are translation factors that distinguish them from those found in the cytoplasm (ribosomes, tRNAs, etc.). ) Has. Electron microscopy, like mitochondria, has a highly organized internal ultrastructure of the chloroplast, which includes:
Includes a flat membrane body known as a lamella or thylakoid disc. However, chloroplasts are typically much larger than mitochondria; in higher plants, chloroplasts are generally cylindrical in morphology, about 5-10 μ in length, and 0.5-2 μ in diameter. Is the range. Similar to mitochondria, which are more abundant in certain tissues (eg, liver) than others, chloroplasts have a higher copy number in some tissues than others. For example, mature leaves contain many chloroplasts, and the total amount of chloroplast DNA in such leaves is approximately twice the total amount of nuclear DNA (Jope et al., J. C.
ell. Biol. 79: 631-636, 1978).

Nucleus and Nucleolar The nucleus is the organelle (mitochondria and chlorophyll) that contains most of the cellular DNA (in terms of information, not mass) in the form of several chromosomes. The body has its own DNA molecules, which are typically much smaller than the nuclear genome, and thus encode fewer functions; however, cells contain only one nucleus and many , And thus the total amount of DNA molecules in these organelles can approach the total amount of nuclear DNA). The nucleus is bounded by two membranes, collectively known as the nuclear envelope, which are known as the inner and outer nuclear membranes. Macromolecules (most specifically RNA
Molecule) is transported to and from the cytosol through an opening in the nuclear membrane called the nuclear pore.

The nucleolus is a small compartment of the nucleus. In contrast to the rest of the nucleus, where messenger (mRNA) molecules are transcribed from DNA, what is produced in the nucleolus appears to be predominantly ribosomal RNA (rRNA) molecules.

(Endosomes, Lysosomes, and Peroxisomes) Cells absorb extracellular fluid and macromolecules dissolved therein by a process called endocytosis. Endocytotic vesicles evolve from a cup-shaped surface feature of a portion of the cell membrane into inwardly directed "buds" and ultimately small membrane-bound vesicles that are taken up by the cytosol. At least two mechanisms have been proposed for the formation of cup-shaped surface features from which endosomes originate. In the first mechanism, the structure of the lipid bilayer portion of the cell membrane and / or
Alternatively, local changes in composition may cause the curvature of the membrane over its limited area. In the second mechanism, one or more coat proteins can act in place in the cell membrane to induce the formation of cup-shaped surface features. In the latter case, the best characterized example is the “coated pit” formed at least in part by the protein clathrin (for review, Schekamn and Or.
ci, Science 271: 1526-1533, 1966).
.

Lysosomes contain various hydrolases, each of which catalyze the degradation of specific types of macromolecules. Primary lysosomes containing such enzymes can be produced intracellularly and fused with endosomes to form secondary lysosomes. In the latter type of vesicles, the enzyme from the primary lysosome comes into contact with the endosome's contents and is therefore free to act on the endosome's contents. Typically,
After enzymatic digestion of the contents of secondary lysosomes, the membrane dissolves to release its contents into the cytosol.

For example, the formation and fate of secondary lysosomes can be advanced using the methods of the invention in the following manner. The cells are engineered to produce one or more lysosomal enzymes that have been modified to contain moieties that can act as acceptors or donors during energy transfer. Such cells come into contact with factors that are taken up by endosomes, where the factors have been or have been modified to be ET acceptors or ET donors, respectively. When the resulting endosome fuses with the primary lysosome, the acceptor and donor are in the same intracellular compartment (secondary lysosome) and ET occurs or can be monitored as described herein. Lysis of secondary lysosomes liberates molecules of the ET acceptor-donor pair, which then separate from each other as they are diluted into the cytosol.
Here the degree of ET is reduced or interrupted together.

Peroxisomes are another type of intracellular vesicle bounded by a monolayer.
Unlike lysosomes, which generally contain hydrolases, peroxisomes contain oxidases that produce and destroy hydrogen peroxide.

Endoplasmic Reticulum The endoplasmic reticulum (ER) is composed of a series of flat sheets, tubes, and sacks surrounding a large intracellular space. The ER membrane is structurally continuous with the outer nuclear membrane and extends throughout the cytoplasm. Some functions of the ER include the synthesis and transport of membrane proteins and lipids. Generally speaking, two types of ER can exist intracellularly. The smooth surface ER is generally tubular shaped and typically has no associated ribosomes; one major function of the smooth surface ER is lipid metabolism. The rough endoplasmic reticulum typically exists as a flat sheet, and many active ribosomes (which perform protein synthesis) are usually bound to its cytosolic side.

Golgi Device The Golgi device is a system of stacked, flat, and membrane-enclosed sacks, and is generally for secretion or delivery to other subcellular compartments. , Is believed to be involved in modification, sorting, and macromolecular packaging. Vesicles surrounded by numerous small (about 50 nM or more) membranes are believed to contain macromolecules to carry out their transport between the Golgi apparatus and other subcellular compartments.

Sub-Organelle Compartments Certain components of organelles are also subcellular compartments within the scope of the present invention. For example, the mitochondria, chloroplast, and nucleus are surrounded by two membranes.
The cavity between the set of paired membranes is not an organelle per se, but it is a subcellular compartment as defined herein. Such a cavity is named, for example, a mitochondrial intermembrane space, a chloroplast intermembrane space, or a nuclear intermembrane space. Such intracavitary conditions and processes are in accordance with the present invention by incorporating a receptor-donor molecule pair transmembranely, or by incorporating a donor or acceptor into the transmembrane space, and acceptor or donating. It can be monitored by incorporating the body into either the intima or adventitia, respectively.

The subcellular compartment may itself be a membrane. In this aspect of the invention, membrane-directed donors (eg, 9-anthrylvinyl (LAPC)) and acceptors (eg, 3-perylenoyl (LPPC)) are incorporated into one or more selected membranes.
The partition coefficient between the membrane and the aqueous phase is 8.
3 × 10 ⁵ and 10.5 × 10 ⁵ (Razinkov et al., Biochim.
． Biophys. Acta 1329: 149-158, 1997).

Intracellular parasites Other subcellular compartments of interest include intracellular parasites such as viruses and intracellular bacteria such as Rickettsiae and Chlamydia spp.
)). The virus has a genome (enclosed by a protein shell, D
Can consist of either NA or RNA). In the case of animal viruses, the protein shell itself is often enclosed within an envelope that contains both protein and lipid. The virus grows only in cells. This is because viruses depend on the host cell's macromolecular synthesis process.
Therefore, they are described as "genetic parasites".

One example of how the present invention may be applied to such intracellular parasites (provided by way of illustration and not limitation) is as follows. Virus particles
It typically consists of a "coat" or capsid that surrounds one or more nucleic acids. The capsid, which typically comprises one or more structural polypeptides, protects the viral nucleic acid in the extracellular milieu, but (if the viral nucleic acid is to be released and replicated) the virus is After being captured, it must be removed. The process by which the capsid is removed is called "uncoating" and is typically the cytoplasm (or subcellular compartments within the cytoplasm (eg, vesicles)).
) Occurs in. Most animal viruses experience uncoating as a result of the action of intracellular proteases on the polypeptides that are part of the capsid. Factors that inhibit or block viral uncoating, for example, by inhibiting the action of intracellular proteases, are expected to be novel antiviral factors, and methods for assaying viral uncoating are described for such factors. Useful for screening.

The invention provides, for example, methods for assaying viral uncoating in the following manner. Viral particles containing an acceptor-donor molecule pair (“loaded virus”) are prepared; eg, donating viral particles that specifically localize to lipid membranes, or cells infected with the virus. This can be achieved by contacting the body-receptor molecule pair. By way of example, and not limitation, the donor can be 9-anthrylvinyl (LAPC) and the acceptor can be 3-perirenoyl (LPPC) (Razinkov et al., Biochim. Biop.
hys. Acta 1329: 149-158, 1997).

Viral adsorption typically occurs equally well at 4 ° C. and 37 ° C., while uncoating proceeds rapidly at 37 ° C., but slowly at 4 ° C. if at all. And proceed. Therefore, the loaded virus is contacted with the cells at 4 ° C for a time allowing complete adsorption, after which the temperature is raised to 37 ° C to allow uncoating to proceed. As the uncoating of the loaded virus progresses, donor-acceptor molecules are released from the capsid, thus they lose their proximity to each other. This loss of proximity results in an increase in fluorescence (when one molecule quenches the fluorescence of the other) or a decrease (when fluorescence is produced when the donor-acceptor molecules are in close proximity to each other). Will be reflected in either. Thus, the rate of change in fluorescence correlates with viral uncoating. Factors that inhibit viral uncoating when added to this assay system reduce or eliminate changes in fluorescence.

Rickettsia is a small, polymorphic, Gram-negative coccobacillus adapted for intracellular growth in arthropods and other organisms. R. Except for quintana, the causative agent of trench fever, all Rickettsiae require viable cells for growth. Species differ in terms of location of intracellular growth; tsusugam
Ushi typically grows only in the cytoplasm, spot heat group organisms proliferate in both the cytoplasm and nucleus, and C. burnetii grows in the cytoplasm and phagolysic corpuscles.

The Chlamydiaceae is a family of obligate intracellular bacterial parasites that infects many vertebrate hosts, typically birds or mammals, including humans. Chlamy
The unique developmental cycle of dia begins with binding to the host cell by the elementary body (metabolic, extracellular phase of Chlamydia) and internalization by this host cell. Phagocytosed elementary bodies develop into a reticular body that proliferates by mitosis. The progeny of the elementary body are formed from replicated reticulate bodies and are released when the host cell ruptures.

The life cycle of Chlamydia represents another non-limiting example of how the present invention can be applied to intracellular parasites. Chlamydia live intracellularly within the phagosome.
Because, in part, this basal cell wall appears to inhibit phagosome fusion with lysosomes, which contain hydrolytic enzymes that degrade the basal bodies when phagosomes are formed. Because it is done. When the elementary bodies are labeled with a donor or acceptor molecule, respectively, and the lysosomes are labeled with an acceptor or donor molecule, energy transfer occurs when phagosomes are formed. Factors that inhibit the ability of the elementary bodies to interfere with the fusion of phagosomes and lysosomes provide energy transfer that can be monitored by the present invention; such factors are novel antibiotics useful for treating Chlamydia infections. Is expected to be.

Assay for Interactions between Macromolecules in or Associated with Subcellular Compartments In another aspect of the invention, energy transfer is found in or binds to subcellular compartments. Used to monitor the interaction between pairs of macromolecules. This embodiment, which relates to a means for monitoring the relationship between macromolecular species and organelles or other subcellular compartments, describes a system in which energy transfer is used to assess the interaction between two types of macromolecules. It should not be confused.

As one example, some cancer cells are believed to result, at least in part, from overexpression of a protein that can be preferentially bound to one or more subcellular compartments. bcl-2
The gene was first identified as the causative agent in certain types of lymphoid cancer (hence the name B-cell lymphoma). Where bcl-2 is overexpressed and B
It is thought to result in an abnormal prolongation of lifespan for the cells, which then accumulates additional mutations that result in overt malignancy and lymphoid neoplasia (Bc).
For a review of the 1-2 family of proteins, see Davies, Trends
in Neuroscience 18: 355-358, 1995; Kroe.
mer, Nature Med. 3: 614-620, 1997; WO95 / 1.
3292; WO95 / 00160; and U.S. Pat. No. 5,015,568).

The biochemical function of Bcl-2 is unknown (ie, it is unclear whether it acts as an enzyme, receptor or signaling molecule), but the outer mitochondrial membrane, nuclear membrane and endoplasmic reticulum It is known to be localized in. Bcl-
Bax, another member of the two family of proteins, is localized to the outer mitochondrial membrane. FRET has been used to demonstrate the interaction of Bcl-2 and Bax in individual mitochondria (Mahajan et al. Nat. Biot.
echnol. 16: 547-552, 1988), energy transfer has not been used to monitor the binding of (or dissociation from) such proteins to subcellular compartments. The present invention provides a method for monitoring the interaction of macromolecules with subcellular compartments.

One example of such a method is as follows. The total width of the inner and outer mitochondrial membranes was estimated to be 22 ± 4 nm (Perkins
Et al., J. Structural Biol. 119: 260-272, 1997.
). Thus, loading the donor (or acceptor) molecules into the transmembrane space is very well enough with the acceptor (or donor) molecules present in or bound to the mitochondrial outer membrane. Expected to be in close proximity. Thus, events such as the localization of the Bcl-2 protein to the outer mitochondrial membrane are associated with Bcl- at the acceptor (or donor) that undergoes energy transfer by the donor (or acceptor) loaded in the transmembrane space. Can be monitored by tagging 2. In this manner, the separation of proteins (eg, cytochromes) from mitochondria can be followed using donor or acceptor-tagged cytochrome c proteins and transmembrane spaces loaded with acceptor or donor, respectively. . Such processes are thought to represent key events in the apoptotic pathway (Green et al., Science).
281: 1309-1312, 1998; Green, Cell 94:
695-698, 1998).

Screening for Species-Specific Agents In certain embodiments, the present invention provides screening assays for identifying species-specific agents. “Species-specific drug” refers to a drug that affects the subcellular compartments of a first organism belonging to one species, but does not affect the homologous subcellular compartments of a second organism belonging to another species. . Thus, the present invention preferentially favors the cell membrane potential in the subcellular compartment of the first biological source, while substantially not altering the corresponding cell membrane potential in the subcellular compartment of the second cytological source. A method for identifying a drug that changes In a preferred embodiment, the subcellular compartment is mitochondria and the cell membrane potential is the mitochondrial membrane potential. Thus, the screening assay provided by the present method involves a relevant portion of assaying cell membrane potential in the absence and presence of a candidate agent, which assay comprises a first and second distinct biological Contacting a first and a second sample, each containing one or more cell membranes from a source, with an ET donor and an ET acceptor molecule, exciting the ET donor to produce an excited ET donor molecule, ET By detecting the signal produced by the energy transfer from the donor to the ET acceptor and comparing the signal produced in the absence of the candidate agent with the signal produced in the presence of the candidate agent.

The present invention preferentially alters mitochondrial membrane potential in mitochondria from a first biological source without substantially altering mitochondrial membrane potential in mitochondria from a second biological source. In a particular preferred embodiment of the method for identifying an agent that does, neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and each of the ET donor and ET acceptor is described herein. As provided therein, independently of each other, they are localized at the same or receptively adjacent sub-mitochondrial sites. Typically, based on the techniques provided herein, one of skill in the art will detect when a candidate agent alters a cell membrane potential, such as the mitochondrial membrane potential, eg, in the absence of the agent. Can be readily determined by detecting a statistically significant change in membrane potential in the presence of this drug, relative to the potential applied. Methods for determining mitochondrial membrane potential are also provided in US patent application Ser. No. 09 / 161,172.

As used herein, mitochondria in mitochondria identified according to the method (either a species-specific agent or from a second biological source (eg, a second species)) identified in the mitochondria A drug that “preferentially” alters mitochondrial membrane potential in mitochondria from a first biological source (eg, a first species) without substantially altering the membrane potential is said to be Mitochondria or cells from one of the species (ie, either the first species or the second species but not both) after contact with the mitochondria or cells of the species Of the mitochondria or cells from the other species, while providing continued viability of the. Similarly, such agents are
Of the species of the first species and the second species after contact with the mitochondria or cells of the species (ie, the first species or Agents that provide continued viability of mitochondria or cells from one of the second species, but not both, while providing killing or impaired proliferation of mitochondria or cells from the other species. Say. Therefore, the preferential alteration of mitochondrial membrane potential by such agents is
As long as this effect is species-specific, ΔΨ _m can be increased or decreased. Without wishing to be bound by theory, cells that experience death or impaired proliferation in a species-specific manner as a result of contact with such agents identified according to the present method become apoptotic or necrotic. It may be experienced by becoming, arresting the cell cycle, or becoming cytostatic, or being unable to survive or grow by any other mechanism.

[0129] In certain other embodiments, the mitochondrial membrane potential in mitochondria from a second biological sample (eg, a second tissue) is not substantially altered and the first biological sample (eg, An agent identified according to the present method that “preferentially” alters the mitochondrial membrane potential in mitochondria from a first tissue) of the first and second biological samples. And then provide continued viability of mitochondria or cells from one of the samples (ie, either the first tissue sample or the second tissue sample, but not both), while the other Refers to an agent that provides for death or proliferation of mitochondria or cells from a sample. Similarly, if such an agent does not “substantially” alter the mitochondrial membrane potential in the mitochondria of the first sample, it may be exposed to the mitochondria or cells of the first and second species after contact with the mitochondria or cells of the first and second species. Provide continued viability of mitochondria or cells from one of the samples (ie, either the first sample or the second sample, but not both), while deriving from the other sample (species) Refers to agents that provide for mitochondrial or cell death or impaired proliferation. Therefore, the preferential alteration of mitochondrial membrane potential by such agents may increase or decrease ΔΨ _m , as long as this effect is sample specific. According to these embodiments, agents that act selectively in a tissue-specific manner can be identified such that they engineer mitochondrial membrane potential in specific tissue types,
Even in the same organism, it can be used to prevent manipulation with other types.
Alternatively, the first tissue and the second tissue may be from separate subjects of the same species or may be from subjects of different species. For example, according to such methods of the invention, agents that preferentially alter neuronal cell mitochondrial potential without substantially altering hepatocyte mitochondrial membrane potential can be identified using this approach.

Using mitochondria as an example of a subcellular compartment, this embodiment of the invention
For example, mitochondria from different species (eg, trypanosomes (As
hkenazi et al., Science 281: 1305-1308, 1998).
As well as other eukaryotic pathogens and parasites, including but not limited to insects, that induce a plunge of ΔΨ, but plunge of ΔΨ in mitochondria found in cells of their mammalian hosts. Can be used to identify agents that do not induce. Such agents are expected to be useful for the prophylactic or therapeutic treatment of such pathogens and parasites.

For example, members of the phylum Apicomplex (formerly known as Sporozoa) include a large and diverse group of pathogenic protozoa that are intracellular parasites. Some members (including Babesia, Theileria and Eimeria species) cause animal diseases of commercial importance, and other members (eg Toxoplasma gondii and Cryptosporidiu).
m spp. ) Also causes human disease, especially in immunocompromised individuals. This acomplexan is unusual for its extrachromosomal DNA element. Because they contain both the mitochondrial and putative plastid genomes (for review, F
eagin, Ann. Rev. Microbiol. 48: 81-104, 19
94). Preferably, the most well-studied Apicomplex (
acomplexan) is a Plasmodium species that causes malaria. As an antimalarial drug, a drug that specifically impairs the function of Plasmodium mitochondria (Peters et al., Ann. Trop. Med. Pars
itol. 78: 567-579, 1984; Basco et al., J. Am. Eukary
ot. Microbiol. 41: 179-183, 1994), and one such drug, atovaquone, is
A sudden drop in ΔΨ in mitochondria from odium yoelii
It has no effect on mammalian mitochondrial ΔΨ (Srivast
ava et al. Biol. Chem. 272: 3961-966, 1997).
. Therefore, a ΔΨ ET-based assay of the present invention screens a library of compounds for novel antimalarial agents (ie, compounds that cause a ΔΨ plunge in Plasmodium-derived mitochondria but not mammalian mitochondria). Can be used for.

As another example, this embodiment of the invention may be directed to unwanted plants (eg, weeds).
Agents that selectively induce a ΔΨ plunge in the mitochondria from which it is desirable but not in plants (eg, cereals) or undesired insects (especially,
It is used to generate and identify agents that selectively induce a ΔΨ plunge in the Lepidoptera family and members of other insects that damage cereals, but not in desirable insects (eg, bees) or desirable plants. Such agents are expected to be useful in the control and control of such unwanted plants and insects. Cultured insect cells (eg, Spodoptera fru
Sif9 cell line and Sf21 cell line derived from piperda, and Trich
source of mitochondria in certain such embodiments of the present invention, including the HIGH FIVE ^™ cell lines from Opolisia ni (including these three cell lines are available from InVitrogen, Carlsbad, Calif.). Can be.

In this embodiment of the invention, the subcellular compartment of interest of the first species is loaded with a first donor-acceptor molecule pair that fluoresces at a first wavelength and a second species. The corresponding subcellular compartment from which is loaded with a second donor-acceptor molecule pair that fluoresces at a second wavelength. For example, mitochondria from two different species can be loaded with such donor-acceptor molecule pairs. The two types of loaded mitochondria are placed in a single chamber, and then agents to be tested for their ability to induce MPT in a species-specific manner are also introduced into that chamber. Changes in fluorescence at both the first and second wavelengths are measured over time in a simultaneous fashion. For example, Fluorometris Imaging Plate Rea
a der (FLIPR ^™ ) device (see below) from a first mode (monitoring fluorescence at a first wavelength) to a second mode (monitoring fluorescence at a second wavelength) It can be used to quickly alternate between. The species-specific agent induces MPT in the mitochondria from the first species but not in the mitochondria from the second species, and thus the extent, rate, frequency or change in fluorescence at one wavelength Affects size but not the other.

Screening for Diagnostic and Therapeutic Agents The present invention can be used to develop assays for subcellular conditions or intracellular processes associated with diseases or disorders for a variety of purposes. One purpose is to assist in the diagnosis and prognosis of patients suffering from such diseases and disorders, and to determine whether an individual potentially has a predisposition to develop such diseases and disorders. It is to be. Another object is to treat patients with such diseases and disorders, or those who potentially have a predisposition to develop such diseases and disorders, curative, therapeutic, palliative, recovery. Effect, prevention (p
Screening the collection of compounds for agents that have a reductive, prophylactic, or disease-blocking effect.

Accordingly, the present invention provides methods for identifying agents that alter cell membrane potentials, and in certain preferred embodiments, methods for identifying agents that alter mitochondrial membrane potentials. In another particular preferred embodiment, the invention provides a method for identifying a regulator of a drug that alters mitochondrial membrane potential. Thus, the screening assay provided by this method relates to assaying the cell membrane potential in the absence or presence of a candidate agent or candidate regulator in the appropriate portion, the assay comprising one or more cell membranes. Contacting the sample with an ET donor and an ET acceptor molecule,
Exciting the ET donor to produce an excited ET donor molecule, detecting a signal resulting from energy transfer from the ET donor to the ET acceptor,
And comparing the signal generated in the absence of the candidate agent (or regulator) with the signal generated in the presence of the candidate agent (or regulator). Embodiments relating to methods for identifying regulators of agents that alter mitochondrial membrane potential include prior to the step of detecting known agents that alter mitochondrial membrane potential or altering mitochondrial membrane potential and provided herein. Further comprising contacting the sample with an agent that is any of the agents identified by the methods described above.

In certain preferred embodiments, the invention relates to methods for identifying agents that alter mitochondrial membrane potential or for identifying regulators of agents that alter mitochondrial membrane potential. In certain preferred embodiments, the ET donor molecule is also The ET acceptor molecule is also not mitochondrial endogenous, and each of the ET donor and ET acceptor is independent of one another at the same sub-mitochondrial site or is acceptably adjacent as provided herein. It is localized in the sub-mitochondrial region. Typically, based on the teachings provided herein, one of skill in the art can readily determine when a candidate agent alters a cell membrane potential (eg, mitochondrial membrane potential), which can be, for example,
By detecting a statistically significant change in membrane potential in the presence of the drug as compared to the potential detected in the absence of the drug. Methods for determining mitochondrial membrane potential are also provided in US patent application Ser. No. 09 / 161,172.

[0137]   Similarly, drug candidates that alter cell membrane potential (eg, mitochondrial membrane potential)
To quantify the membrane potential to determine whether a compound is a regulator
The method is useful. Drugs that change the mitochondrial membrane potential include
Drugs known to have properties (drugs and mitochondria that consume mitochondrial membrane potential)
Drugs that cause a sudden drop in the membrane potential of the Tochondria (see, for example, in more detail in the Examples below).
Ed.), As well as identified by the methods provided herein.
Drugs may be mentioned. With regulators of drugs that change the mitochondrial membrane potential
In certain ways, agents that modify the mitochondrial membrane potential are mitocon
Directly or indirectly affect the ability to change the doria membrane potential (eg increase or increase).
Or reducing) any agent. Thus, for example, mitochondria
The regulator of the drug that alters the membrane potential can be an agonist, or mitco
It may be an antagonist of a drug that alters the Ndria membrane potential. For example, Mitocon
When a drug that modifies the doria membrane potential consumes that potential, it is an agonist, regi.
A curator may enhance such consumption (eg, cause a plunge), while
, A regulator that is an antagonist of drugs that alter the mitochondrial membrane potential
Protects against the mitochondrial membrane potential in the presence of the drug it consumes.
Can give fruit. Conversely, ΔΨ_mMitochondria by storing or enhancing
For drugs that alter the membrane potential, regulators that are agonists also
Protects or enhances, while the antagonist regulator _m Can result in consumption or plunge. I don't want to be bound by theory
Regulators as described in the book modify mitochondrial membrane potential
Subcellular targets or targets of drugs and drugs that alter mitochondrial membrane potential (mi
Tochondria molecular components (for example, mitochondrial molecular components are described in US Patent Application No. 0
Intermolecular interaction with one or more of
Action event (eg, recognition, binding, complex formation, covalent modification, conformational change)
Can be involved in.

Thus, when numerous disorders and diseases result from processes involving mitochondria, the main energy source in cells in higher organisms, the present invention, as described above, mitochondrial membrane potential (ΔΨ) and energy transfer. Compositions and methods for monitoring mediated changes in mitochondrial membrane potential are provided. As described herein, ΔΨ requires a variety of mitochondrial functions, and defects in the production or maintenance of ΔΨ are associated with many diseases and disorders. Moreover, changes in ΔΨ occur in various subcellular processes that can serve as targets for the development of therapeutic agents. Therefore, ET-based assays for ΔΨ help to identify the presence of a disease or disorder associated with alterations in ΔΨ in an individual, or the predisposition of the individual to such disease or disorder,
And to screen for agents that (appropriately) stabilize, increase or decrease ΔΨ and therefore can be used to treat such diseases and disorders,
Can be used. In addition, ΔΨ ET-based assays screen for agents that selectively perturb ΔΨ in unwanted cells (eg, cancer cells), resulting in the specific disruption or inhibition of proliferation of such unwanted cells. Can be used to

Mitochondria provide direct and indirect biochemical regulation of respiratory, oxidative and metabolic processes in a wide range of cells (for review Ernster and S.
chatz, J .; Cell Biol. 91: 227s-255s, 1981), such regulation involves electron transport chain (ETC) activity, which produces metabolic energy in the formation of adenosine triphosphate (ATP). Drives the oxidative phosphorylation of A. and underlies a central role of mitochondria in intracellular calcium homeostasis. In addition to its role in metabolic processes, mitochondria are also involved in suicide progression of genetically programmed cells, known as "apoptosis" (Green and Reed, Sci.
ence 281: 1309-1312, 1998, Susin et al., Bioch.
em. et Biophys. Acta 1366: 151-165, 199.
8).

Defective mitochondrial activity, including, but not limited to, defects in the elaborate multi-complex mitochondrial assembly known as the electron transport chain (ETC) at any stage can result in (i) A decrease in ATP production, (ii) an increase in the production of highly reactive free radicals such as superoxide, peroxynitrite and hydroxy radicals, and hydrogen peroxide, (iii)
3.) Disorders in intracellular calcium homeostasis and (iv) release of factors that initiate or stimulate the apoptotic cascade (eg cytochrome c and "apoptosis inducing factor"). Due to these biochemical changes, mitochondrial dysfunction can cause extensive damage to cells and tissues.

A number of diseases and disorders are believed to be caused by or associated with inappropriate induction or suppression of mitochondrial-related functions that result in alterations in mitochondrial metabolism and / or apoptosis. These include, but are not limited to, the following: chronic neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD); autoimmune diseases; diabetes mellitus (including types I and II). ); Mitochondrial related diseases (congenital muscular dystrophy with mitochondrial structural abnormalities, lethal infantile myopathy with severe mtDNA deletion and benign "with moderate loss of mtDNA").
"Late-onset" myopathy, MELAS (mitochondrial encephalopathy, lactic acidosis, and seizures) and MIDD (mitochondrial diabetes and hearing loss); MERFF (myoclonus epileptic red rag fiber syndrome (
myoclonic epilepsy ragged red fiber
Syndrome)); arthritis; NARP (neuropathy; ataxia; retinitis pigmentosa); MNGIE (myopathy and external ophthalmoplegia; neuropathy; gastrointestinal; brain disorders), LHON (Laver hereditary optic neuropathy); Pearson disease; PEO (progressive external ophthalmoplegia); Wolfram syndrome; DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, deafness); Leigh syndrome; ataxia; schizophrenia; and hyperproliferative disorders (For example, cancer, tumors and psoriasis).

According to the generally accepted theory of mitochondrial function, proper ETC respiratory activity requires maintenance of the electrochemical potential (ΔΨ) in the inner mitochondrial membrane by the coupled chemo-osmotic theory. Conditions for consuming or plunging this membrane potential (ETC
), Including but not limited to defects at all stages of, can thereby interfere with ATP biosynthesis and interfere with or stop the production of biochemical vital energy sources. Altered or defective mitochondrial activity is also referred to as "mitochondrial permeability alteration" (MPT), a catastrophic mitochondrial collapse (coll).
apse) can occur. In addition, mitochondrial proteins (eg, cytochrome c and “apoptosis inducing factor”) can be separated from or released from mitochondria due to MPT (or the action of mitochondrial proteins such as Bax), and Proteases known as caspases can be induced and / or stimulate other events in apoptosis (
Murphy, Drug Dev. Res. 46: 18-25, 1999).

Defective mitochondrial activity may alternatively, or additionally, result in the production of highly reactive free radicals with the ability to damage cells and tissues. These free radicals can include reactive oxygen species (ROS) (eg, superoxide, peroxynitrite and hydroxy radicals) and other potentially reactive species that can be toxic to cells. For example, oxygen free radical-induced lipid peroxidation is a well-established pathogenic mechanism in central nervous system (CNS) injury (eg, found in many degenerative diseases and ischemia (ie, stroke)). is there. Mitochondrial involvement in the apoptotic cascade (eg, mitochondrial release of cytochrome c) has been identified and thus may be a factor in neuronal death that contributes to the pathogenesis of certain neurodegenerative (ie, CNS) diseases.

In addition, there are at least two deleterious consequences for exposure to reactive free radicals resulting from mitochondrial dysfunction, which deleteriously affect the mitochondria themselves. First, free radical-mediated damage is one of ETC's various proteins.
One or more can be inactivated. Second, free radical-mediated damage is associated with "permeability changes (
catastrophic disruption of mitochondria, termed "transition permeability". According to the generally accepted theory of mitochondrial function, proper ETC respiratory activity requires maintenance of the electrochemical potential at the inner mitochondrial membrane by a coupled chemo-osmotic mechanism. Free radical oxidative activity can consume this membrane potential, thereby impeding ATP biosynthesis, and / or triggering mitochondrial events in the apoptotic cascade. Thus, by modulating these and other effects of free radical oxidation on mitochondrial structure and function, the present invention protects mitochondria not provided by the mere determination of free radical-induced lipid peroxidation. Compositions and methods.

For example, rapid changes in mitochondrial permeability are likely to be accompanied by changes in the inner mitochondrial transmembrane protein adenylate translocase, which results in the formation of “pores”. It is not clear whether this pore is a clear conduit in the membrane or is simply a widespread leak. At a minimum, permeability changes are enhanced by exposure to free radicals, so permeability changes are more likely to occur in mitochondria of cells from patients with mitochondrial-related disorders that are chronically exposed to such reactive free radicals. Can be.

The altered mitochondrial functional properties of disease-associated midchondria may also be associated with loss of mitochondrial membrane electrochemical potential by mechanisms other than free radical oxidation, and such alterations in permeability may be associated with mitochondrial genes, It may result from the direct or indirect effects of the gene product or associated downstream mediator molecule and / or extra-mitochondrial gene, gene product or associated downstream mediator, or from other known or unknown causes.

Diabetes mellitus Diabetes mellitus is a common degenerative disease, affecting 5-10% of the population in developed countries. The propensity to cause diabetes mellitus is reportedly maternally inherited and the involvement of mitochondrial genes has been proposed (Alcolado et al. Br. Med. J.
． 302: 1178-1180, 1991; Reny, Int. J. Epide
m. 23: 886-890, 1994). Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are very concordant, and among the first associations of affected individuals, there is a high frequency of disease.

At this level of cells, a degenerative phenotype that may be characteristic of late-onset early diabetes mellitus is the altered mitochondrial respiratory function (eg, impaired insulin secretion, decreased ATP synthesis, and levels of reactive oxygen species). Increase) inheritance. Studies have shown that diabetes mellitus can be developed or associated with certain associated disorders. For example, it is estimated that 40 million individuals in the United States suffer from late onset impaired glucose tolerance (IGT). Patients with IGT do not respond to glucose with increased insulin secretion. Small proportion of IGT individuals (5
Insulin deficient non-insulin dependent diabetes mellitus (NIDDM)
Develop into. Some of these individuals have insulin-dependent diabetes mellitus (IDD).
Further develop into M). These forms of diabetes mellitus (NIDDM and IDDM
) Is associated with decreased insulin release by pancreatic β-cells and / or decreased end organ response to insulin. Other symptoms and conditions of diabetes mellitus that precede or are associated with diabetes mellitus include obesity, vascular disease, peripheral neuropathy, sensory neuropathy, blindness and deafness.

Due to the strong genetic component of diabetes mellitus, nuclear genes have been the focus of research into the causative gene mutations. However, despite diligent efforts, the nuclear genes that segregate diabetes mellitus are known only to mutations in the rat in the insulin gene, insulin reporter gene, adenosine deaminase gene and glucokinase gene. Thus, mitochondrial defects, including but not limited to defects associated with individual non-nuclear mitochondrial genomes present in mitochondrial DNA, can contribute significantly to the pathology of diabetes mellitus (
Anderson, Drug Dev. Res. 46; 67-79, 1999).
.

Multiple mitochondrial mutations associated with the diabetic phenotype have been described (for review, Gerbitz et al., Biochim. Acta 1271: 253-2.
60, 1995 or Rotig et al., Diabetes Metab. 22: 2
91-298, 1996). Multiple such mutations are associated with gene coding factors (eg, mitochondrial t
RNA) (eg, Suzuki et al., Diabetes Car).
e 17: 1428-1432, 1994; Kishimoto et al., Diabe.
tologia 38: 193-200, 1995; van der Ouwe.
land et al., Muscle Nerve Suppl. 3: S124-S130
Hanna, et al., Am. J. Hum. Genet. 56: 1026-
1033, 1995; Sano et al. Neurol. 243: 441-444
, 1996; Kameoka et al., Biochem. Biophys. Res. C
ommun. 245: 523-527, 1998; and Hirai et al. C
lin. Endocrinol. Metab. 83: 992-994, 1998
checking). Mitochondrial migration is described by ΔΨ (Cote et al., J. Biol.
Chem. 264: 8487-8490, 1989; Cote et al., J. Am. Biol
． Chem. 265: 7532-7538, 1990), a modification of ΔΨ may be obtained in the diabetic phenotype in some cases, and in individuals suspected of having developed diabetes, or in developing diabetes. Suspected individuals can be identified using the ET-based assay ΔΨ of the present invention. Further, a reagent that increases and / or stabilizes ΔΨ is effective in treating patients with developing diabetes or those who are considered to have developing diabetes to have corrective, therapeutic, temporary palliative, rehabilitative, Preventative effect, preventive (p
It is expected to have a phylactic effect or a disease-blocking effect. The ΔΨ ET-based assays of the present invention can also be used to estimate which reagents are most effective for a given individual, patients with mitochondria exhibiting modified ΔΨ are associated with normal ΔΨ. It is expected to be more responsive to agents that regulate ΔΨ than patients with mitochondria.

Parkinson's Disease Parkinson's disease (PD) is a substantia nigranopa of the brain.
is a progressive, chronic mitochondrial-related neurodegenerative disorder characterized by loss and / or atrophy of dopamine-containing neurons in rs compacta. PD, like Alzheimer's disease (AD), also afflicts the elderly. P
D is characterized by bradykinesia (slow movement), rigidity and resting tremor. L-Dopa treatment temporarily suppresses tremor in most patients, but ultimately, this tremor becomes less controllable and can be felt by the patient on his own or in his own hygiene. Make it difficult or even impossible to meet your needs.

The neurotoxic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson's syndrome in animals and humans at least in part via mitochondrial effects It is shown. MPTP is converted to active metabolite (MPP +) in dopamine neurons;
It is concentrated in mitochondria. This MPP + then selectively inhibits the mitochondrial enzyme NADH: ubiquinone oxidoreductase (“Complex I”), increasing free radical production, decreasing adenosine triphosphate production, and ultimately developing dopamine neurons. Lead to death.

Apoptotic cell death is thought to constitute a terminal process in several neurodegenerative diseases, particularly Alzheimer's disease and Parkinson's disease. It has been proposed that reagents that help maintain ΔΨ may provide novel reagents for preventing or treating neurodegenerative apoptosis (Tatton et al., Ann. Neuro.
l. 44: S134-S141, 1998). Developing Parkinson's disease (PD)
Individuals suspected of having developed or developing Parkinson's disease can be identified using the ET-based assay ΔΨ of the present invention. In addition, the ET-based ΔΨ assay of the present invention is used to identify and characterize compounds that enhance or stabilize ΔΨ, and these compounds, once a patient suffers from developing PD or becomes developing PD. For possible patients, corrective, therapeutic, temporary palliative, rehabilitative, preventative, preventative (
It is expected to have a prophylactic effect or a disease blocking effect. The ΔΨ ET-based assay of the present invention can also be used to estimate which reagents are most effective for a given individual, and PD patients with mitochondria exhibiting modified ΔΨ will obtain normal ΔΨ. Δ more than PD patients with associated mitochondria
It is expected to be more responsive to reagents that regulate Ψ.

Alzheimer's Disease Alzheimer's Disease (AD) is a chronic, progressive neurodegenerative disease characterized by neural deficit and / or atrophy in individual areas of the brain, extracellular deposition of β-amyloid and cellular. Is associated with the accumulation of neurofibrillary tangles within. AD is a disease unique to humans, and it occurs in more than 13 million people worldwide. AD also
It is a unique and tragic disease. Many individuals, who live a normal productive life, are gradually attacked by AD as they age, and the disease is gradually deprived of the patient's memory and other intellectual abilities. Ultimately, this AD patient becomes unaware of his family and loved ones, and often requires ongoing care until his death.

Mitochondrial dysfunction is believed to be important in the cascade of events leading to apoptosis of various cell types (Kroemer et al., FASEB J.
． 9: 1277-1287, 1995), and may be responsible for apoptotic cell death in neurons of the AD brain. Modified mitochondrial physiology may be among the earliest events in PCD (Zamzami et al., J. Exp. Med. 182:
367-77, 1995; Zamzami et al., J. Am. Exp. Med. 181: 1
661-72, 1995), high levels of reactive oxygen species (ROS) resulting from such modified mitochondrial function may initiate the apoptotic cascade (
Auserer et al., Mol. Cell. Biol. 14: 5032-42, 1
994). In particular, the predominant pathology of AD is the death of selected neuronal populations in individual areas of the brain. Cell death in AD is presumed to be apoptotic. Because the signal of programmed cell death (PCD) has been elucidated, and no indicator of active gliosis and necrosis has been found (Smale et al. Exp. Neurolog. 133: 225-230, 1995). ; Cotma
n et al., Molec. Neurobiol. 10: 19-45, 1995). AD
The consequences of cell death, neuronal loss, and synaptic loss in E. coli are closely associated with the clinical diagnosis of AD and are highly associated with the degree of dementia of AD (DeKosky et al., Ann. Neurology). 2757-464, 19
90).

In some cell types, including neurons, the mitochondrial membrane potential (Δ
The decrease in ψ) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, a fraction enriched in mitochondria but not in the nucleus can induce nuclear apoptosis (Newmeyer et al., Cell 70: 353-.
64, 1994). Moreover, cytoplasmic hybrids containing mitochondria derived from AD patients have lower resting mitochondrial membrane potentials than the corresponding parental SH-SY5Y cell line, and cyclosporin A reverses the reduced ΔΨ in AD cytoplasmic hybrids ( Cassarino et al., Biochem. Biophys. Res.
． Commun. 248-173, 1998). Individuals suspected of having developed AD, or suspected of having developing AD, can be identified using the ET-based assay ΔΨ of the present invention. In addition, the ET-based ΔΨ assay of the present invention is used to identify and characterize compounds that enhance or stabilize ΔΨ, and these compounds are identified as patients suffering from or developing AD. Corrective, therapeutic, temporary palliative, rehabilitative, preventive, prophylactic effects in potential patients
It is expected to have an effect or a disease blocking effect. The ΔΨ ET-based assay of the present invention can also be used to estimate which reagents are most effective for a given individual, and AD patients with mitochondria exhibiting modified ΔΨ will have normal ΔΨ.
It is expected to be more responsive to reagents that regulate ΔΨ than AD patients with mitochondria with.

Other Neurological Disorders Similar theories have been developed for similar relationships between mitochondrial defects and other neurological disorders, which include Alzheimer's disease, Leber's hereditary optic nerve. Disorders, schizophrenia, "mitochondrial encephalopathy, lactic acidosis, and seizures (MELAS)" and "myoclonus epilepsy Raggat red muscle fiber syndrome (MERRF)".

Increased evidence indicates a functional role for mitochondrial dysfunction in chronic neurodegenerative diseases (Beal, Biochim. Biophys. Acta 13
66: 211-223, 1998), and recent studies have involved mitochondria to regulate events leading to necrotic and apoptotic cell death (S).
usin et al., Biochim. Biophys. Acta 1366: 151-
168, 1998). Stressed (eg, especially by free radicals, high amounts of intracellular calcium, lack of ATP, etc.) mitochondria release pre-formed lytic factors that initiate apoptosis via interactions with apoptosomes. Yes (Marchetti et al., Cancer
Res. 56: 2033-2038, 1996; Li et al., Cell 91:47.
9-489, 1997). Release of preformed lytic factors (eg, cytochrome c) by stressed mitochondria occurs as a result of a number of events. In any event, the magnitude of stress (ROS, intracellular calcium levels, etc.) affects changes in mitochondrial pathology that ultimately determine whether cell death occurs via the necrotic or apoptotic pathways. it is conceivable that. To the extent that apoptotic cell death is a hallmark of degenerative disease, mitochondrial dysfunction can be an important factor in disease progression. To the extent that the ΔΨ inhibition or plunge is a causative or compounding factor in degenerative diseases, individuals suspected of having developed such a disease during development, or individuals suspected of having such a disease during development, are of the present invention. It can be identified using the ET-based assay ΔΨ. The ET-based assays of the present invention are used to identify and characterize reagents that enhance or stabilize ΔΨ, and these reagents are used in patients suffering from or developing such diseases. Corrective, therapeutic, temporary palliative, rehabilitative, preventative (preventive)
) Effect, prophylactic effect or disease blocking effect. The ΔΨ ET-based assays of the present invention can also be used to estimate which reagents are most effective for a given individual, and patients with mitochondria exhibiting altered ΔΨ will have normal ΔΨ. It is expected to be more responsive to agents that regulate ΔΨ than patients with mitochondria with.

Stroke In contrast to chronic neurodegenerative disease, neuronal death following a stroke occurs in an acute fashion. A large body of literature now demonstrates the importance of mitochondrial function in neuronal death following ischemia / reperfusion injury associated with stroke, heart failure and trauma to the brain. Experimental support continues, with alterations in mitochondrial function leading to incomplete energy metabolism, increased oxygen radical production and impaired intracellular calcium homeostasis, and involvement of active mitochondria in the apoptotic cascade in the pathology of acute neurodegeneration. The central role is accumulated.

Strokes occur when perfusion is reduced in areas of the brain, and neurons die in an acute or delayed fashion as a result of sudden ischemic events. The cessation of blood supply to the brain reduces the tissue's ATP concentration to negligible levels within minutes. In the center of infarction, deficiency in mitochondrial ATP production results in deficient ionic homeostasis leading to osmotic cytolysis and neuronal death. Multiple secondary changes may also contribute to cell death after reduction of mitochondrial ATP. Cell death in acute neuronal injury extends from the center of the infarction, where the neurons first die by necrosis, to the border where the neurons undergo apoptosis, and to the periphery where tissue is damaged (Martin et al., Brain Res. Bull. 46: 281-
309, 1998).

Most of the neuronal damage in the border zone was observed during cytolysis in infarct lesions (
Especially when aggravated by lack of bioenergetics in mitochondria due to lack of oxygen)
Caused by excitotoxicity induced by glutamate released into the brain (MacMa
nus and Linnik, J .; Cerebral Blood Flow M
etab. 17: 815-832, 1997). The first attraction in excitotoxicity is primarily the large influx of Ca ²⁺ through the NMDA receptor, which results in increased Ca ²⁺ uptake into mitochondria (Cell Death, reviewed by Dykens). and Diseases of the Ne
rvous System, V.I. E. Koliatos and R.K. R. Data
n. Humana Press, New Jersey, pages 45-68, 19
99 "Free radicals and mitochondrial d
ysfunction in excitotoxicity and neu
rode generative diseases "). Ca ²⁺ overload is
It lowers the mitochondrial membrane potential (ΔΨ _m ) and induces increased production of reactive oxygen species (Dykens, J. Neurochem 63: 584-591, 19).
94; Dykens, The Oxygen Paradox, K .; J. A. D
avies and F.I. Ursini ed., Clear Press, U. of P
adova, pp. 453-467 (1995), "Mitochondrial.
radical production and mechanisms o
f oxidative exoticity "). Cytochrome c and other proteins where severe enough ΔΨ _m plummet and mitochondrial Ca ²⁺ sequestration can induce pore opening in the mitochondrial membrane through a process called mitochondrial permeability change (MPT). Releases (initiates apoptosis) indirectly (Bernardi et al., J Biol Chem 267).
: 2934-2939, 1994; Zoratti et al., Biochim Bio.
Phys Acta 1241: 139-176, 1995; Ellerby et al., J Neurosci 17: 6165-6178, 1997). Consistent with these observations, glutamate-induced excitotoxicity can be inhibited by interfering with mitochondrial Ca ²⁺ uptake or by blocking MPT (Budd et al., J. Neurochem 66: 403-411, 1996; White. ,
J. Neurosci 16: 5688-5697, 1996; Li et al., Bra.
in Res 753: 133-140, 1997; Stout et al., Nat. N
eurosci. 1: 366-373, 1998).

Agents and methods that maintain mitochondrial integrity during transient ischemia and subsequent waves of excitotoxicity are novel neuroprotective agents with utility in limiting seizure-related nerve injury. Is expected. Given the limitations of the therapeutic window for inhibition of necrosis in the infarct core, developing therapeutic strategies to prevent neuronal death by preventing mitochondrial dysfunction in the non-necrotic region of the infarction. Limiting may be particularly desirable. As described in more detail herein in Example 9, such agents have been tested for their ability to stabilize ΔΨ under excitatory conditions that mimic transient ischemia. It can be isolated by screening the population. Such agents may provide salvageable, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-blocking disease to patients who have, or who are considered susceptible to, seizures. Expected to have an effect. The ΔΨ ET-based assays of the invention can also be used to assess which agents are most likely to be effective in a given individual. That is, patients with mitochondria that show changes in ΔΨ are more likely than patients with mitochondria with normal ΔΨ to
It is expected to be more likely to respond to drugs that regulate ΔΨ.

Hyperproliferative Disorders Although mitochondrial-mediated apoptosis may be important in degenerative diseases,
Disorders such as cancer are thought to involve unregulated and unwanted growth of cells (hyperproliferation). Such cells somehow escape the mechanism that normally triggers apoptosis in such unwanted cells. Enhanced expression of the anti-apoptotic protein Bcl-2 and its homologues has been implicated in many human cancer pathologies. Bcl-2 is programmed for cell death and Bc
Protein B, which acts by inhibiting overexpression of 1-2 and is related
cl-X _L is blocked mitochondrial release of cytochrome c, and the activation of caspase 3 from mitochondria (Yang et al., Science 275
: 1129-1132, 1997; Kluck et al., Science 275: 1.
132-1136, 1997; Kharbanda et al., Proc. Natl. A
cad. Sci. U. S. A. 94: 6939-6942, 1997). In contrast, overexpression of Bcl-2 and Bcl-X _L is protected against mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. further,
Is a acquired multidrug resistance to cytotoxic drugs is associated with inhibition of Chitokutomu c release that is dependent on overexpression of Bcl-X _L (Kojima et al., J.Biol.C
hem. 273: 16647-16650, 1998).

Inhibit the growth of cells and tissues that avoid appropriate apoptotic signals, or
Or there is a need for compounds and methods that enhance its death, as well as cytotoxic agents that cause the death of unwanted cells (eg, cancer cells) by inducing an apoptotic cascade or other methods. In particular, mitochondria are mediators of apoptotic events, so agents that stimulate mitochondrial-mediated pro-apoptotic events are particularly useful. Since mitochondria are involved in apoptosis, agents that interact with mitochondrial components (contents) are expected to bring about the ability of cells to undergo apoptosis. Such agents are salvageable, therapeutic, palliative, rehabilitative, preventative in patients with or thought to be prone to hyperproliferative disorders (eg, cancer and psoriasis). It is expected to have a prophylactic, preventative or disease-disturbing effect. The ΔΨ ET-based assays of the invention can also be used to assess which agents are most likely to be effective in a given individual. That is, ΔΨ
It is expected that patients with mitochondria exhibiting changes in A will be more likely to respond to agents that regulate ΔP than those with normal mitochondria.

The mitochondrial ΔΨ ET-based assays of the present invention can also be used to identify agents that are selectively cytotoxic to hyperproliferative cell types or other unwanted cell types. For example, ΔΨ is elevated in some carcinoma cell lines, and agents that accumulate in mitochondria as a function of ΔΨ (eg, rhodamine 123) are preferentially cytotoxic to such carcinoma cells (Mod.
ica-Napolitano et al., Cancer Res. 47: 4361-4
365, 1987; Andrews et al., Cancer Res. 52: 1895
~ 1901, 1992).

In summary, the present invention relates to subcellular conditions or intracellular processes (eg, changes in mitochondrial ΔΨ) for identifying and characterizing agents that treat degenerative disorders and diseases as well as hyperproliferative disorders. Can be used to develop an assay for The ΔΨ ET-based assay can be used to identify agents that are mitochondrial protective agents, anti-apoptotic agents or pro-apoptotic agents, depending on the disease or disorder for which treatment is sought.

The following examples illustrate the invention and are not intended to limit the invention. One of ordinary skill in the art, through routine experimentation, will be able to recognize or identify many equivalents to the particular materials and procedures described herein. Such equivalents are considered to be within the scope of this invention.

EXAMPLES Example 1 Selection of compounds and optimization of conditions for ET-based assays Subcellular fractions (subcellular compartments) (eg organelles or organelles) To detect conditions within its membrane-bound portion) and to develop ET-based assays to monitor the changes, determine the appropriate pair of donor and acceptor compounds, and their useful concentrations. It is necessary. Using the information and methods provided herein, one of skill in the art can readily determine suitable donor and acceptor compounds, and their concentrations, for a variety of such assays.

One step in the process of developing an ET-based assay involves optimizing the concentration of donor and acceptor compounds, as well as other conditions for this assay. Generally, at least two criteria apply regarding the concentrations of donor and acceptor compounds. First, the concentration of donor and acceptor compounds must be sufficient for energy transfer to occur. Second, the concentration of each compound is (a) minimal of any non-ET based signal from this compound, resulting in minimal background signal in this assay, and (b) cells Any undesired effects on physiology, including cytotoxicity, and / or effects on the subcellular fraction of interest should be low enough to be minimal. However, it should be noted that not all compounds have undesired effects on cell physiology.

For the FRET-based assay of ΔΨ using NAO and TMRM, these criteria apply as follows. NAO has a higher concentration (eg Maf
> 10 μM, as reported by tah et al. (FEBS Lett. 260: 236-240, 1990), is known to be toxic to certain cells, so the NAO sensitivity of the cells to be used is Must first be determined to avoid exposing toxic levels of this ET molecule. N is toxic
AO concentration depends on the cell (eg, cell line) selected for use in a given experiment, and other conditions (eg, exposure time to NAO, presence of other virulence or protective factors, etc.) And can change. However, one of ordinary skill in the art can select and readily determine the appropriate NAO concentration and / or time of NAO exposure to cells, without undue experimentation, based on the present disclosure.

[0171] For example, a series of experiments were performed to demonstrate that for a FRET-based assay of ΔΨ,
Optimal ratios and concentrations of NAO and TMRM were determined. These tests
It can be applied to other pairs of donor and acceptor compounds in other ET-based assays.

General Protocol A FRET-based assay for ΔΨ was generally performed in the following manner. However, modifications to these general procedures can be made without affecting the sensitivity, accuracy or efficiency of the assay.

Cell Lines and Their Preparation Various cell lines were used in the following experiments. Neuroblastoma SH-SY5Y is a multiple subcloned cell line of human origin (Perez-Polo et al., De.
v. Neurosci. 5: 418-423, 1982). SH-SY5Y is
It is a well-characterized cell line that can differentiate into neuron-like cells and is an accepted cell model for various neuronal cell functions (for a review see, eg, Vaughan et al., Gen. Pharmacol. 26: 1191).
~ 1201, 1995; Pahlman et al., Acta Physiol. Sca
nd. Suppl. 592: 25-37, 1990).

[0174] Cybrid (cytoplasmic hybrid) cells contain nuclear components from one cell type and cytoplasmic components from another cell type (including mitochondria). Procedures for the preparation of cytoplasmic hybrid cells, derived from mitochondrial DNA (mtDNA) depleted cells (rho ⁰ or ρ ⁰ ) and containing mitochondria from patients with Alzheimer's disease, have been previously described (Miller Et al., J. Neurochem. 67: 1897-1907, 1996; Swed.
Low et al., Neurology 49: 918-925, 1997; and US Pat. No. 5,888,498, all of which are incorporated herein by reference. The 1685 cytoplasmic hybrid cell line is one of this type of cytoplasmic hybrid cell line.
Here is an example. The 1685 cytoplasmic hybrid cell line was created by fusing SH-SY5Y neuroblastoma cells that had rho ⁰ created by long-term treatment with ethidium bromide with platelets from the AD donor. "MixCon" is a similar fashion, but using platelets from n normal, age-matched patients (n = 2-3 depending on the particular experiment) for the construction of cytoplasmic hybrid cells. Shows a mixed control (Mixed Control) composed of prepared cytoplasmic hybrids.

NCI-H460 is registered under the registration number ATCC HTB-177 in America.
n Type Culture Collection (ATCC, Manas
sas, VA) is a human lung large cell carcinoma cell line. NCI-H460
A preferred cell culture medium for cells is 90% (2 mM L-glutamine, 1.5 g /
L sodium carbonate, 4.5 g / L glucose, 10 mM HEPES and 1.
RPMI 160 medium containing 0 mM sodium pyruvate), 10% (fetal bovine serum).

MCF-7 is a human breast cancer cell available under the accession number ATCC HTB-22 from the ATCC. MCF-7 has been used in studies of the relationship between mitochondrial ΔΨ collapse and apoptotic events (eg Heer.
dt et al., Cancer Res. 59: 1584-1591, 1999). A preferred cell culture medium for MCF-7 cells is 90% MEM (Minimum Essential Eagle Medium supplemented with 2 mM L-Glutamine and Earl BSS, 1.5 g.
/ L sodium carbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate), 10% FBS-ins (fetal bovine serum containing 10 μg / ml bovine insulin).

In general, cells are plated in 96-well microplates (Costar, black wall;
Plated on clear, flat bottom) with about 2-3 × 10 ⁴ cells per well at least about 24 hours prior to assay. HBSS is commonly used as the cell culture medium, but any medium suitable for a given cell line can be used in the assay.

(Preparation of Donor Compound and Acceptor Compound) ET Donor Compound (Nonyl Acridine Orange) (NAO, Molecular)
r Probes, Inc. , Eugene, OR; Catalog No. A1372)
A 5 mg / ml stock solution of was prepared in DEMSO. Aliquot this stock solution into a microcentrifuge tube and thaw on ice just prior to assay,
It was stored frozen at -20 ° C. 5 for use in assays unless otherwise specified
The NAO stock solution of mg / ml was added to Hanks balanced salt solution (HBSS, Lif).
e Technologies, Grand Island, NY)
Diluted to 1: 5000 to produce a working stock solution containing 1 μg / ml NAO. This was further diluted as shown below.

Stock solutions of receptor compounds (100 mM TMRM (Molecular
Probes, Inc. , Eugene, OR; catalog number T668), D
Prepared in MSO. This concentration corresponds to 20,000 x final concentration (used in the assay). This solution was aliquoted into microcentrifuge tubes and stored frozen at -20 ° C until thawed on ice just prior to assay.

Combined stock solutions were also prepared to facilitate handling. This solution was prepared in DMSO with both ET donor and acceptor compounds (25 mM).
TMRM and 1 mg / ml NAO) (ie both 5,000 times the final concentration of ET molecules used in the assay). This combined stock solution was aliquoted into microcentrifuge tubes and stored frozen at -20 ° C and thawed on ice just prior to assay.

Instrument Preparation The FLIPR ^™ heater and laser were turned on for at least 1 hour before performing the assay. The following settings were typically used: shutter, 0.4 sec; f-stop, 0.2; filter, # 2; 300 mW (15 A) laser. In a later experiment, a custom filter for 530 ± 25 nm (Omega O
optical, Inc. , Brattleboro, VT) was used.

On the FLIPR ^™ instrument there are positions for three 96-well microplates. The centrally located 96-well microplate contains the sample,
And up to two 96-well plates containing additional reagents (one on each side of the central plate) can be included. In a typical assay, a 96-well (8 rows, 12 columns) plate of the first reagent was set up so that the wells in row A had medium (
Typically containing HBSS), and the wells in row B contain ΔΨ plunging agents (typically CC
CP), and the remaining columns (CH) contained test compounds (eg, candidate drug). In addition, in a Type II assay (see Example 5), a second reagent plate was set up so that each well had an appropriate amount of ΔΨ plunging agent that was occasionally added to the sample after the test compound. (Typically CCCP).

Loading of Fluorophores Fifteen minutes prior to the assay, the entire plate was flushed and gently flipped over (flic).
k) The cell culture medium was removed. Replaced medium with 5 μM TMR in each well
Replaced with 100 μl HBSS containing M and preheated to 37 ° C. In general,
It was preferred to preheat the medium and reagents to 37 ° C. and maintain the cells at 37 ° C. to avoid heat shock which could itself cause changes in ΔΨ or cause death of susceptible cells. After 10 minutes, 20 μl of NAO working stock solution (1 μg / ml NAO) prepared as described above was added to each well (final concentration, 200 ng / ml, equal to 0.4 μM).

In an optional step, after allowing the cells to incubate in the presence of both fluorophores for about 5 minutes, gently flip the plate to remove the cell culture medium and 100 μl of preheated HBSS. Excess fluorophore was removed by adding to each well; this process was repeated up to 3 times. Finally, after inverting the plate, 100 μl of preheated HBSS was added to each well. Depending on the cell type used in a particular experiment, the cells can be treated without apparent loss of ET signal prior to addition of test compound (eg, candidate drug).
It can be incubated for various periods of time. In the case of SH-SY5Y cells, this incubation period was up to about 40 minutes.

Assay Readings Approximately 20 readings were taken on the FLIPR instrument at 3 second intervals prior to the addition of test compound. Although these data are not used to calculate the results of the assay, they are useful for assessing cell integrity and / or for monitoring the spontaneous collapse of ΔΨ. Is. For example, when compromising cell integrity, a significant plunge in ΔΨ is detected after the optional rinse step but prior to the addition of the test compound. The test compound was then added and 175 readings were taken at 5 second intervals.

To determine the ET value that corresponds to the largest plunge in ΔΨ (ie, ΔΨ˜0 in theory), a ΔΨ plunging agent (eg, CCCP) was added as follows. In the Type I assay (FIG. 3A), the depressant was added to the wells different from the wells receiving the test compound at about the same time that the test compound was added, and these wells were read for the test compound. Reads were taken at the same time that the wells received were read. In the Type II assay (FIG. 3B), readings were taken at 5 second intervals for a period of time (typically about 9-15 minutes), followed by the addition of a depressant to the same wells receiving the test compound, and then reading. Was carried out for a second period, which was approximately equal to the first period.

Data Analysis The results of the assay were analyzed for qualitative analysis using fluorescence units relative to time (RFU).
) As a plot (FIG. 6). For quantitative analysis, the calculations were as follows: For Type I assays, the first instrument reading for each well was adjusted to zero. Reads made at 5 second intervals (typically the reads numbered from about read 21 to about read 195-200) followed by reads taken at 3 second intervals to confirm cell integrity. Totaled (ΣF _x ). Significance test for multiple (ie, greater than 2) groups (eg, untransformed treatment group one-way A
NOVA (one-way ANOVA), (Newman-Keuls or
Bonferroni (Dunn's) multiple comparisons)) was used to assess the significance of the results.

For the Type II assay, adjust the first instrument reading of each well to zero,
Then, readings (about 2 times) taken at 5 second intervals (after integrity check, as described above).
1 to about 195-200) were summed (ΣF _x ). Average the readings during the last 4 minutes (ie, read numbers of about 214-230) after addition of ΔΨ plunging agent (CCCP) to maximize compromise of membrane potential for normalization. _Yes (F _CCP ). Since the use of ratios violates the mathematical assumptions specific to the ANOVA algorithm, the data were transformed (log or arcsine) before being evaluated for significance in a one-way ANOVA analysis.

For each type of assay, the sum and average for each well was calculated as F
Calculated using the software provided with the LIPR ^™ instrument. ^* . EXCEL ^™ (Microsoft, Inc., Redmond, WA) via txt
Was sent to. And about ANOVA, GB Stat (Dynamic Mi)
CROSS SYSTEMS, Silver Springs, MD). FLIPR ^™ kinetic data is sent to EXCEL for the calculation of the mean and standard error of readings taken over time. It is desirable (but not necessary) to back up all FLIPR ^™ data to a CD, or another suitable machine-readable format on a daily basis.

Results In the first set of experiments MixCon cytoplasmic hybrid cells were loaded with TMRM (0, 1.25, 2.5, 5.0, 10 and 20 μM in 96 well plates).
) And NAO (0, 6, 12, 25, 50, 100, 200 and 400 ng)
/ Ml; 0, 13, 26, 52, 105, 210, 420 and 840, respectively.
nM) at 6 different concentrations. Each of the 48 possible combinations of TMRM and NAO concentrations was 0.1% with a laser set at 300 mW.
Duplicate shutters were used to test in duplicate with the FLIPR instrument and readings were taken using a 510-590 nm filter. CCCP was added to all samples (1.5 μM) 1 minute after placing the plate on the FLIPR ^™ instrument.

According to a non-limiting theory, when FRET occurs between NAO and TMRM, which are located in the inner mitochondrial membrane and mitochondrial matrix, respectively, the change in FRET-based signal is due to CCCP addition. Should happen later. Therefore, the addition of CCCP reduces ΔΨ and, consequently, the mitochondrial concentration of the receptor compound (TMRM). Because
TMRMs excite and / or are absorbed by mitochondria to a lesser extent. Since the mitochondria retain the donor compound (NAO) regardless of ΔΨ, the donor compound and the acceptor compound are FR
The ETs are no longer in close proximity to each other to occur and the signal resulting from FRET should be reduced, as indicated by the change in fluorescence (expressed in relative fluorescence units (RFU)).

Also, according to a non-limiting theory, the energy transfer from NAO to TMRM is
It can be measured either directly or indirectly (see Figure 1). NAO → T
A direct measurement of MRM FRET is the appropriate wavelength [λD (ex
)], Exciting a donor (NAO), which then emits energy at a wavelength [λD (em)] that overlaps with the excitation spectrum of the acceptor (TMPM), and (b) was excited. Measuring the emission from the TMRM molecule at or near its peak emission wavelength [λA (em)]. N
The indirect measurement of AO → TMRM FRET also involves exciting NAO with λDex, but the emission from the donor NAO is measured rather than the emission from the acceptor TMRM (ie, λA (em)). Rather λD (em) is measured). When energy transfer efficiently occurs from the excited donor (NAO) to the acceptor (TMRM), the emission from the donor is “quenched” and λD (em)
The signal at is minimized. When the acceptor compound is no longer in close proximity to the donor, energy transfer no longer occurs and the emission from the donor is "dequenched (d
“equipped” (ie, the signal at λD (em) is
To increase).

In this example, FRRT was measured indirectly. TMRM + NAO loaded on SY5Y cells was irradiated with light having a wavelength of 488 nm (λD (ex) (4) for NAO).
(Near 85 nm) and 530 ± 25 nm (λD (NAO for NAO
The signal in the vicinity of (em)) was measured over time after the addition of CCCP. FR
If ET occurs between the donor NAO and the acceptor TMRM, the addition of CCCP, which results in a decreased concentration of TMRM in mitochondria, should result in dequenching of the signal from NAO ( That is, increasing fluorescence at or near λDem) is expected. In contrast, FR
When ET is measured directly, the signal at or near λD (em) for TMRM is measured, and the signal is expected to decrease after addition of CCCP and consequent release of TMRM from mitochondria. It

The results of the indirect FRET measurement are shown in FIG. In these results, FRET was found as an increase in the signal, the dequenching of the emission of NAO, which indicates that the addition of CCCP was only present when both donor and acceptor compounds were present in the concentration of the given combination. Occurred later (ie, this increase is
It did not occur when either the acceptor compound or the donor compound was present alone at the same concentration). For example, in FIG. 4, FRET shows wells A9 and A1.
Occurred in wells E9, E10, F9, F10, 9G and 10G compared to signals in 0 (absence of NAO) and wells F1 and F2 (absence of TMRM). Although FRET was probably occurring in the other wells in the extreme right hand portion of Figure 4, the signals in these wells were also NAO alone (eg comparing wells H11 and H12 with H1 and H2). Or T
MRM alone (eg compare H11 and H12 with A11 and A12)
It may include a significant background signal component from the source. Based on these results, a useful and useful concentration of NAO and TMRM for the assay is 5
0 ng / ml NAO and 10 μM TMRM (wells E9 and E10),
100 ng / ml NAO and 10 μM TMRM (wells G9 and G10
), And 200 ng / ml NAO and 5 μM TMR (wells G7 and G8).

The data in FIG. 4 was analyzed as described above for the Type I assay (ie, the first reading for each well was set to 0 and about 21 to about 1).
The RFU readings taken at 75 seconds were summed (ΣF _x ). The results for various concentrations of TMRM were graphed as a function of NAO concentration, as shown in FIG. FRET occurred at 50, 100 and 200 ng / ml NAO containing 5 or 10 μM TMRM, as evidenced by the increase in signal at these concentrations (eg in the range of 50-200 ng / ml NAO). Curves of 5 and 10 μM TMRM were analyzed for 0,
(Compare with 1.25 and 2.5 μM TMRM curves).

The background signal of each fluorophore was individually as well as CCCP mediated Δ
In another experiment designed to investigate the time course of a Ψ plunge, FRET was
More prolonged period after CCP addition (1.5 μM), 5 μM TMRM, 420
It was measured in cells treated with either nM NAO or both compounds. As shown in FIG. 6, the rapid increase in fluorescence is due to CCCP
It occurred within the first 2 minutes after the addition, after which the change in fluorescence reached a plateau. N
The fluorescent signal was essentially constant when either AO or TMRM were present alone.

The following experiments were performed to determine if the NAO dequenching signal measured in the FRET-based assay was linear over different cell densities. MixCon cells or 1685 cytoplasmic hybrid cells with different numbers of about 1
Pre-incubated in wells of a 96-well plate containing 5 μM TMRM for 0 min, then 4 ng / ml NAO was added and cells were incubated for an additional 5 min. Finally, CCCP (1 μM) was added to each well and the fluorescent signal was 530 ± 2 using the FLIPR ^™ device.
Monitored at 5 nm. Mitochondrial efflux of TMRM then occurred, as evidenced by an increase in fluorescent signal corresponding to the dequenching of NAO radiation over time. The first slope of the curve (RFU versus time) was plotted against the number of cells per well. The result is that the ΔΨ-dependent fluorescent signal is
It was shown to increase in a linear fashion for the range of about 38,000 to about 330,000 cells per well.

Although many of the experiments described herein use the FLIPR instrument and involve a series of measurements over time, the present invention was performed using any instrument or device of sufficient sensitivity. It may be possible to monitor at least two time points (ie, before and after the addition of agents that affect ΔΨ). In one experiment, for example, MixCon cells and 1685 cytoplasmic hybrid cells were preincubated with TMRM and NAO as described above, and fluorescence at 538 nm was measured using fmax ^™ (Molecular Devices, Inc., S.
(univale, CA) was measured using a fluorimetric plate reader (excitation = 485 nm) and then treated with CCCP (final concentration, 1.3 μM). 1
After 0 minutes, the fluorescence at 538 nm was measured again and all three cell types (SY
5Y cells, MixCon cytoplasmic hybrids, 1685 cytoplasmic hybrids) were found to be significantly increased when compared to control cells treated with buffer alone.

In addition, 1685 cytoplasmic hybrid cell lines, including mitochondria from patients with Alzheimer's disease, are more sensitive to ionomycin (ie, control cytoplasmic hybrid cells (MixCon) or parental SH-SY5Y cells). Indicates a greater degree of loss of Δψ than the strain). This result uses the assay
We show that differences between cell types can be detected in the response to agents that affect Δψ.

Example 2 Parameter-dependent co-localization of acceptor-donor compounds Detect conditions within subcellular compartments (eg, organelle or its membrane-bound proteins) and monitor changes in them. Another step in the process of developing an ET-based assay for is not only the co-localization of donor and acceptor compounds sufficiently close for energy transfer to occur, but also such co-localization. To make sure that the localization depends on the state of the measured parameter. That is, at least one compound, as a function of the measured parameter,
Must be localized (accumulated) in the subcellular compartment of interest, and remain in that compartment,
And / or if the parameter changes, it should not accumulate too quickly or efficiently in that compartment.

For example, for an ET-based assay designed to measure mitochondrial Δψ, one of the compounds (either donor or acceptor) accumulates in mitochondria in a Δψ-dependent manner, and The presence of the other compound in the mitochondria (receptor or donor, respectively), which must be released from the mitochondria, must be Δψ-independent. Combining these criteria with the information provided herein, one of skill in the art can readily select an appropriate donor-acceptor combination for an ET-based Δψ assay.

Compounds whose mitochondrial concentration is independent of Δψ include, but are not limited to, the following: NAO (Petit et al., Eur. J. Bio.
chem. 194: 389-397, 1990; Maftah et al., Bioche.
m. Biophys. Res. Comm. 164: 185-190, 1989).
, MitoTracker® Green FM (US Pat. No. 5,45).
9,268 and 5,686,261), MitoFliuor ^™ G
reen (Haugland, Handbook of Fluorescen
t Probes and Research Chemicals, 6th Edition,
Spence ed., Molecular Probes, inc. , Eugene
, Oregon, 1996, 269), and (a) red-shifted or yellow-shifted green fluorescent protein polypeptides, or "FLASH" polypeptides,
And (b) a fusion protein comprising a polypeptide sequence that localizes the fusion protein to the mitochondrial matrix or inner membrane. These compounds are listed in Table 2 as I
Listed as Group V and Group V donor compounds. A series of representative Group IV and V receptor compounds are also shown in Table 2. Accumulation in mitochondria in the group IV and group V receptor compounds of Table 2 in a Δψ-dependent manner includes, but is not limited to, the following: rhodamine 123 (Emau).
s et al., Biochem, Biophys. Acta 850: 436-448,
1986; Scaduto et al., Biophys. J. 76: 469-477: 1
999), TMRM and TMRE (Farkas et al., Biophys. J. 5).
6: 1053-1069, 1989; Ehrenberg et al., Biophys.
J. 53: 785-794, 1988).

Regarding specific sites of accumulation of these compounds, NAO interacts specifically with the inner mitochondrial membrane (Maftah et al., Biochem. Biophys.
Res. Comm. 164: 185-190, 1989). Without wishing to be bound by theory, TMRM, TMRE and Rhodamine 123 are thought to localize to the mitochondrial matrix, but a recent report has shown that these compounds are also associated with internal and external mitochondrial membranes. In surface, we show reversible accumulation, probably as a function of the localized microenvironment, which is characterized by enhanced membrane potential (Scaduto et al., Biophys. J. 76: 469.
-477, 1999). Therefore, regardless of whether TMRM, TMRE and rhodamine 123 localize to the inner mitochondrial membrane or the mitochondrial matrix (or both), they are expected to reside closer to the inner mitochondrial membrane, NAO is localized in the inner membrane (Fig. 1
checking).

The following experiments were performed to confirm that only FRET occurred between properly localized donor-acceptor pairs of compounds in living cells. SH-SY5Y cells were cultured and assayed as in Example 1 with the following exceptions.
Cells were incubated with 5 μM “acceptor” compound for 10 minutes and then further incubated with 4 ng / ml “donor” compound for another 10 minutes. At this time, a drug (CCCP) that rapidly decreases Δψ was added to cells at a concentration of 1 μM, and relative fluorescence was measured by fmax ^MT (molecular Device).
es, Inc, Sunnyvale, CA) Fluorometric plate reader (excitation,
485 nm; emission read at 538 nm ± 20 nm). 6
The average rate of change of Relative Fluorescence Units (RFU) in ~ 8 replicate wells as the slope of the curve over the first 3.5 minutes using software provided on the fmax ^MT instrument via least squares linear regression. I calculated.

The results are shown in Table 4. FRET detected between NAO and TMRM, C
They localize to the inner mitochondrial membrane and mitochondrial matrix, as indicated by the average rate of FRU change after the addition of CCP. FRET occurred between NAO and TMRM until CCCP was added, which caused a decrease in Δψ and release of the receptor compound (TMRM) from mitochondria. Since the donor compound (NAO) is retained by mitochondria independently of Δψ, the donor and acceptor compounds cease to exist sufficiently close to each other for FRET to occur, and the signal is , FRET decline (which is indicated by the relatively rapid rate of change of RFU).

[0206] In contrast to the effects seen with NAO and TMRM, calcein or CO-.
When Fluor was used as the "donor" compound, the rate of RFU change following the addition of CCCP was negligible. This means that calcein and CO-Fluo
Although r has emission peaks similar to those of NAO, they do not localize to mitochondria and therefore the "receptor" compound (TMRM localized to mitochondria) is sufficient for FRET to occur. Reflects the fact that they were not close. In a similar fashion, when SNAFL (not localized to mitochondria) is used as the "acceptor" compound and NAO is used as the "donor" compound:
Even if the SNAFL excitation peak wavelength (514 nm) is closer to the NAO emission peak wavelength (517 nm) than the TMRM excitation peak wavelength (544 nm), FRET was not observed. Therefore, as expected, energy transfer required spectral overlap and physical proximity.

[0207]

[Table 4] In summary, energy transfer (FRET in this example) occurs only if the ET donor and acceptor molecules are properly co-localized within the subcellular compartment of interest.
occured. Furthermore, the process of localizing different pairs of ET donor or ET acceptor molecules in a manner such that pairs of ET molecules are no longer sufficiently close to cause energy transfer is a result of energy transfer. It was monitored and assayed by measuring the changes in the signal generated.

Example 3 Parameter-Dependent Change in Energy Transfer The previous example shows how to determine energy transfer between an ET donor molecule and an ET acceptor molecule, the donor and the acceptor. How to Optimize ET Assay Conditions Including Concentrations of Somatic Compounds and How to Demonstrate Energy Transfer Depends on Colocalization of Both Compounds within the Same or Adjacent Subcellular Sites Indicates An ET-based assay detects a condition or parameter within a subcellular compartment of interest and tests the assay with an agent that has a known effect on the selected condition or parameter to monitor changes in it. Is useful.

Using FER-based assays designed to measure mitochondrial Δφ as a model, a variety of agents are known in the art to either reduce (consume) or eliminate (blunt) Δφ. ) Is known. Moreover, some agents are known to increase Δφ above normal levels, ie to hyperpolarize mitochondria. Both types of drugs have a Δψ FRET
It was evaluated using a base-based assay.

Drugs that Increase Δψ Oligomycin is an example of a compound that hyperpolarizes mitochondria.
MixCon cytoplasmic hybrid cells were contacted with TMRM (5 μM) and NAO (420 nM) as in Example 1. On the same 96-well plate, a second set of MixCon cells was also treated with 10 μM oligomycin, lysed in HBSS buffer for 10 minutes prior to addition to cells and prior to addition of TMRM. Cells were added for 10 minutes. The “initial FRET signal”, ie the first reading before the onset of the Δφ plunge, was determined using the FLIPR ^™ instrument to detect 3
It was determined for each 8 individual wells of the combination.

According to a non-limiting theory, if a drug acts as expected, hyperpolarization should increase Δψ, increase TMRM accumulation in mitochondria, and then increase energy transfer. Guide (NAO quenching). These results (Table 5) show that oligomycin had the expected effect. That is,
Since oligomycin-treated cells contain hyperpolarized mitochondria, the initial FRET signal is significantly less than that seen in cells not exposed to oligomycin.

[0212]

[Table 5] ^* Calculated by two-tailed t-test (Agents that reduce Δψ and protective agents) (Ionomycin and Boncrekinic acid), alone or in combination with Boncrekinic acid (BKA), which consumes calcium ionophore ionomycin (which causes Δψ to be consumed, Then, the effect of ΔP) of (which causes a final sharp drop) was compared with the effect of the Δφ plunge drug CCCP. BKA is
Since BKA binds to the adenine nucleotide translocator and its activity is required for mitochondrial permeability change (MPT), BKA is improving on the Δψ consumption caused by ionomycin.
It has been shown to have an olating effect. SH-SY5Y cells were treated according to the procedure described in Example 1 with donor and acceptor compounds (NA, respectively).
O, 420 nm, and TMRM, 5 μM), and HBBS medium, CCCP (
1.5 μM), ionomycin (5 μM), or ionomycin (5 μM) and BKA (2 μM; preincubated with cells for 10 min at 37 ° C. before adding TMRM). RFU was monitored using a FLIPR ^™ instrument.

These results (FIG. 7) are similar to those of the previous example in that CCCP (FIG. 7, “C
)) Induces a rapid increase in fluorescence, which is clearly due to the quenching of the NAO emission signal and is consistent with a sharp fall in Δψ and migration of the receptor compound TMRM from mitochondria. Treatment with ionomycin (FIG. 7, “I”) finally resulted in a more modest change in fluorescence, as expected for agents known in the art to cause slower consumption in Δψ than in CCCP. was gotten. Addition of BKA to ionomycin treated cells (FIG. 7, “I +
BKA ") mitigates the effects of the ionomycin effect, and ultimately HBSS.
Medium alone (FIG. 7, “MO”) produced a fluorescent signal similar to that seen when added to cells.

(Ionomycin and Ruthenium Red) It was confirmed that ruthenium red has a protective effect on the Δψ consumption effect of ionomycin. Ionomycin is an ionophore that increases levels of cytosolic calcium; this leads to the consumption of Δψ as the mitochondria take up calcium from the cytosol. Ruthenium red blocks the activity of the mitochondrial calcium uniporter and thus inhibits or blocks mitochondrial calcium uptake. Therefore, ruthenium red is expected to offset the effects of ionomycin. SH-SY5Y cells were prepared and pre-incubated with NAO and TMRM as in the previous example and only with CCCP (1.5 μM), ionomycin (5 μM) with ruthenium red (100 μM) and medium (HBSS). Processed. Fluorescence, F
Measured over time using a LIPR ^™ instrument at 530 ± 25 nm. The results (FIG. 8) show that the FRET-based assay yielded data that followed the expected pattern: ionomycin-mediated consumption of Δψ was essentially completely blocked by ruthenium red. Show.

(Ionomycin or MPP ⁺ and Cyclosporin A) In another related experiment, cyclosporin A was confirmed to have a protective effect on the Δψ consuming effect of ionomycin. Cyclosporin A binds cyclophilin D and, like BKA, is expected to block MTP and thus offset the effects of ionomycin. MixCon cells were prepared and preincubated with NAO and TMRM as in the previous example and treated with ionomycin (5 μM). CCCP one group of cells
Was preincubated with cyclosporin A (10 μM) for 15 minutes prior to the addition of. Fluorescence was measured over time using a FLIPR ^™ instrument at 530 ± 25 nm. The results (FIG. 9) showed that the FRET-based assay yielded data that followed the expected pattern, ie, ionomycin-mediated consumption of Δψ was inhibited by cyclosporin A. In another experiment, using that assay, cyclosporin A (10 μM, added for 10 minutes prior to the addition of Δφ drug) was otherwise used to induce long-term (Δφ) of Δφ caused by 0.5 mM MMP ⁺ . It was found to essentially block consumption and plunge (more than 10 minutes after addition).

Atractyloside and Cyclosporin A The FRET assay in the above and in the previous examples also showed that the assay reached a peak of atractyloside (ATR) at about 6 minutes after adding ATR.
, 5 mM) treated SH-SY5Y cells. At this ATR concentration, Δψ recovered after 15 minutes, but CC
CP (1 μM) led to a further complete plunge of Δφ maintained for at least 15 minutes. Pretreatment with cyclosporin A (5 μM, 5 min) resulted in a significant relaxation of the response to ATR; the peak fluorescence signal in the ATR + cyclosporin A sample was approximately half that of the sample treated with ATR alone. It was

In summary, energy transfer (in this example, FRET) is a function of donor and / or acceptor compounds (in this example, decreasing acceptor compound TM).
The concentration of RM was reduced) occurred in a manner that accurately reflected the changes in the parameters (Δψ in these examples) known to affect the concentration of RM. In addition, the selection parameter (Δψ) is increased (eg oligomycin) or decreased (eg
For example, the measured activity of agents known to be CCCP, ionomycin, MPP ⁺ , ATR) was consistent with their expected effects. The same result,
This applies to protective agents (BKA, ruthenium red, cyclosporin A) which are known to counteract in whole or in part with the effects of parameter-altering agents. These results show that the ET-based assay has a selection parameter (Δψ in this example).
Can be used to screen and evaluate compounds that have not previously been characterized for their effect on, and for their ability to counteract the effect of known compounds on the parameter of interest.

Example 4 Evaluation of Assay Results The results presented in the previous examples demonstrate the need to evaluate the results of ET-based assays in a manner that produces meaningful conclusions. Using the results shown in FIG. 7 as an example, the initial rate of change of RFU of samples treated with CCCP, ionomycin or ionomycin and BKA was similar from about 78 seconds to about 127 seconds, although The readings of the three samples then diverged and were significantly different after 460 seconds. For example, as summarized in Table 6, there are various ways to evaluate the results of ET-based assays using the results shown in FIG.

One way to evaluate an ET-based assay is to measure the time taken for each sample to reach a defined RFU value (ie, determine the blocking threshold for each sample). is there. Such a determination indirectly reflects the initial slope of the curve. However, as shown in Table 6, the selection of an appropriate RFU blocking threshold is important in this evaluation method. For example, RFU as a cutoff amount
Choosing = 2220 yields results that are inconsistent with the expected effects of various treatments on Δψ (ie CCCP>ionomycin> ionomycin and BKA> medium only). Furthermore, since the sample treated with ionomycin and BKA blocks RFU = 2220 twice, the results for RFU = 2220 also show that
Somewhat confusing. On the other hand, choosing a low blocking value (RFU = 160) yields results with the predicted order. However, in the latter case, the protective effect of BKA may not be fully understood, as the result of ionomycin + BKA (0.345) is only slightly different than the protective effect of inomycin alone (0.400). Absent.

Another way to evaluate the ET-based assay is to directly determine the initial slope of the curve for each sample. However, as the results shown in Figure 7 show,
Although the overall shape of the curves and their endpoints are different, data from different samples can yield curves with similar initial slopes.

Another evaluation method is to sum the area under the curve of the plot, or a similar operation (eg adding the RFU value at each time point for each sample over a given time frame). Is the way to do it. As shown in Table 6,
This method results in four treatments consistent with the predicted order of effects on Δψ (ie CCCP>ionomycin> ionomycin and BKA> medium only). Thus, although other evaluation methods may be used, summing the areas under the respective curves, or performing an operation that produces a result corresponding to the area under the curves, is preferred in many instances.

[0222]

[Table 6] Example 5 FRET-Based Δψ Assay The above example was based on the “type I” FRET-based Δψ assay, where ΔΔ.
The effect of various drugs on ψ illustrates the potential limitation of drugs that plunge Δψ (compared to the effects of CCCP) (FIG. 3A). A "Type II" assay was developed to produce more meaningful results. In the Type II assay, the drug being tested is first added to the sample, and some time after allowing the drug being tested to exert their effect, to drive Δφ to 0, Δ
The ψ-dropping agent is subsequently added to the same sample, thus establishing the resulting baseline value.

FIG. 3B shows a Type II assay. In one version of the Type II assay, where the compounds are tested for their ability to consume Δψ, the symbols in FIG. 3B are as follows: The optimal first reading (“A” or “B”) that can be normalized to 0 is taken first. Candidate Δψ consuming compounds are added at time “2”. Candidate Δ
If the ψ consuming compound had little or no effect on Δψ, a signal as predicted by the fixed line “C” would be expected, whereas the test compound consuming Δψ is shown by the dashed line (C). Signal is generated. At time “3”, Δφ is completely
Drug (eg, CCCP) is added, and cell density, and E
To allow normalization of various samples for changes in loading efficiency of T donor and acceptor compounds, the reading (
"D") is taken. The Δψ consumption activity of the test compound is Δψ according to the following formula.
Calculated as the consumption value: Δψ consumption value = (C−B) / (D−B) where a higher Δψ consumption value indicates a higher Δψ consumption capacity of the candidate compound.

In another version of the Type II assay, where the compounds are tested for their ability to inhibit or enhance the activity of the Δψ consuming agent, the symbols in FIG. 3B are as follows: The optimal first reading ("A") that can be normalized to 0 is taken first. Test compounds are added at time "1" and a baseline measurement ("B") is taken. Δψ consumer (eg, ionomycin, atractyloside, etc.)
] Is added. If the test compound had little or no effect on the activity of the Δψ consumer, a signal as indicated by the dashed line (“C”) would be expected, but Δψ
A test compound that inhibits or protects against the activity of a consumer is given a fixed line ("
C ") gives rise to a signal. At time point “3”, a drug that completely plunges Δφ (eg, CCCP) was added, and Δφ was allowed to allow normalization for changes in cell density and efficiency of loading of donor and acceptor compounds. The reading ("D") is taken after the plunge in is complete. The activity of the test compound is calculated as the effect index according to the following formula: Effect index = (C−B) / (D−B) where a lower value of the effect index indicates a higher protective effect of the test compound. Show.

CCCP and ionomycin are used in the following exemplary experiments, although other Δψ-depressants are known and can be used. Such Δψ plunging agents include, by way of example, but not limited to valinomycin, A23187 and 4-Br-A23187.

The conditions for the Type II assay are preferably such that Δφ falls sharply and the measured signal is
Any Δψ plunging agent to reach a plateau in a rapid manner (ie, preferably within 5 minutes, more preferably within 3 minutes, and most preferably within 2 minutes after addition of Δφ plunging agent). Establishing a dose response curve of
desirable. Another parameter that can be established from dose response experiments is the optimal concentration of the Δψ plunging agent.

A curve of dose response to CCCP is shown in FIG. In the experiments performed to generate the data in this figure, SH-SY5Y cells were treated with 420 nM NAO and 5 μM TMRM according to the general procedure of Example 1, and then a designated amount of CCCP was applied. It was monitored for about 60 seconds before being added. The quenching of the emission signal from NAO was measured in the previous example. When higher concentrations of CCCP are used, the dose response curve shows an increasing rapid loss of NAO extinction, as evidenced by an increasing rapid rise in RFU. The response to 10 μM CCCP was slightly greater than that seen when 5 μM CCCP was applied (2.5 μM CCCP and 5 μM CCCP).
(Compared to the change in response between CCCP), these data also show 10
It was suggested that μM was close to the saturating concentration of CCCP used.

Results from a representative Type II FRET experiment are shown in Figure 11 and show the relative fluorescence units ± standard error of readings taken at the indicated time points. In this experiment, SH-S
Y5Y cells were contacted with NAO and TMRM according to the procedure of Example 1 and placed in the FLIPR instrument. After about 2 minutes, half of the samples were treated with pre-warmed medium only, and the other half with 5 μM of Δψ consumer 4-bromo.
-Treat with pre-warmed medium containing A23187. After approximately 6.5 minutes, the Δφ plunging agent CCCP (final concentration, 5 μM) was added to all samples and their fluorescence was measured for an additional 7.5 minutes. As shown in FIG. 11, 4-bromo
Cells treated with −A23187 (“4-BR”) showed a gradual loss of Δφ increase until the time when CCCP was added, at which Δφ was further reduced and finally plunged. . Also, as shown in FIG. 11, cells treated with medium (“MO”) also showed a rapid loss of Δφ after CCCP addition and approached a complete Δφ plunge with MO and 4-BR. The curve became progressive after about 600 seconds and for the rest of the experiment.

Example 6 Dose Response Curves for Δψ Consumers and Δψ Protective Agents In establishing the basic parameters of the ET-based assay for Δψ, a more accurate experiment was conducted in which the assay was It was done to show that it can be used to generate a dose response curve for both Δψ protectors. SH-SY5Y cells were used in these experiments. The calcium ionophore inomycin is
Used as a pseudo-candidate drug compound whose ability to cause consumption of Δψ is evaluated,
And cyclosporin A was used as a pseudoionomycin protectant.

The cells are grown to a particular cell density and, as described above, 96
Transferred to well plate. TMRM and NA for both sets of experiments
O was added at the concentrations and order and times described in Figure 1. In experiments containing only ionomycin, ionomycin was added at various concentrations 10 minutes after addition of NAO. In the case of an experiment designed to quantify cyclosporin A's ability to protect against the effects of ionomycin, cells were washed and exposed to varying concentrations of cyclosporin A prior to beginning instrument reading. After 15 minutes of exposure, cells were loaded for 10 minutes TMRM and 5 minutes NAO as described above for fluorophore (ET donor and acceptor molecule) loading.
Readings numbered 1-21 are recorded at 3 second intervals, and then 22-
Readings numbered 196 were recorded at 5 second intervals. As shown in FIG. 13, the sum of the fluorescence signals over each time interval was determined and the log ₍₁₀₎ ionomycin concentration (M
) Was plotted against.

Dose response curves for cells exposed to ionomycin in three separate experiments (50,000 cells per well in each experiment) are shown in FIG. When plotted, the data produced parallel curves, indicating that assay reproducibility in analyzing compounds has a negative effect on Δψ.

A dose response curve of cells pre-treated with varying amounts of cyclosporin A and then exposed to ionocoicin in 3 separate experiments (39,000 cells per well in each experiment) is shown in FIG. Be done. The data produced a similar curve when plotted, showing the reproducibility of the assay in analyzing compounds that protect mitochondria from drugs with a negative effect on Δψ.

Example 7 FRET in Various Cell Types In the previous example, a FRET-based Δψ assay was produced from a neuroblastoma cell line (SH-SY5Y), as well as ρ ⁰ SY5Y cells. MixC
on and 1685 cytoplasmic hybrid cell lines. Control (MixCo
n) and Alzheimer's disease (1685) cytoplasmic hybrids showed the same general response to various agents and treatments affecting Δψ, with some differences in FR.
Detected by ET-based assay. In this example, MixCon or 1685 cells (approximately 50,000 cells per well) were preincubated with 420 nM NAO and 5 μM TMRM according to the procedure of Example 1,
Then, calcium ionophore A23187 (0-5 μM) was added. NAO
Detectable loss of signal (ie fluorescence at 530 ± 25 nm) was measured over a period of time (4 minutes).

The results are presented as the sum of all data points over the four fine concentration ranges for each concentration of A23187 (FIG. 14), and 1 with SH-SY5Y parental cells.
It reveals some differences between the 685 and MixCon cytoplasmic hybrids. The AD (1685) cytoplasmic hybrid showed the highest degree of sensitivity to A23187, and the control (MixCon) cytoplasmic hybrid was the parent S.
It was somewhat more sensitive to A23187 than H-SY5Y cells.
Statistical analysis (ANOVA) showed that the increased susceptibility of AD (1685) cytoplasmic hybrid cells was significant. Thus, the Δψ ET-based assay of the present invention can be used to characterize mitochondrial abnormalities in whole cells. If such cells are isolated from an individual who has, or is suspected of having, a mitochondrial-related disorder (eg, a disorder associated with altered mitochondrial function), the assay will test for such disorders. It can be used as an aid in diagnosis.

Example 8 ET-Based Assay for Detecting Specific Cell Types in Samples An energy transfer-based assay is used to detect specific cell types in biological samples. obtain. For example, Rhodamine 123 (Group II, III and IV receptor compounds; see Table 2) is rapidly taken up and after washing by various human carcinoma cells for long periods (longer than 24 hours) (e.g. This Rhodamine 123 is maintained (if not always fully maintained by this cell type) when other cell types are washed (Nadakavukaren et al.
al. , Cancer Res. 45: 6093-6099, 1985; Su.
mmerhayes et al. , Proc. Natl. Acad. U. S.
A. 79: 5292-5296, 1982; Christman et al.
, Gynecol. Oncol. 39: 72-79, 1990).

Accordingly, an ET-based assay for carcinoma cells in a sample includes the following steps: (1) obtaining a biological sample from a patient, where:
This sample contains cells (including, for example, carcinoma cells); (2) contacting the cells in this sample with rhodamine 123; (3) washing the cells as needed; (4) The cells were treated with mitochondrial donor compounds from groups II, III or IV (Tables 2 and 3) (eg NAO, MitoTrack).
er (registered trademark) Green FM or MitoFluor ^™ Green)
Contacting with; (5) exciting the sample with light having a wavelength in the excitation spectrum of the donor; and (6) detecting energy transfer as quenching of the donor emission by rhodamine 123. . The carcinoma cells retain Rhodamine 123 and thus show FRET with the donor compound.

The following experiments were performed to perform specific cell types (in this example, human carcinoma cell lines).
Bind and retain specific ET donor and / or acceptor molecules as provided herein differentially, thus such specific cell types are It is shown to have a unique characteristic that allows it to be detected by a base-based assay, thereby distinguishing such cell types from other cell types that may be present. NCI-H460 is a human lung large cell carcinoma cell line (see Example 1 for details). NCI-H460 cells were added to 96 well plates (approximately 50,000 cells per well). TMRM of type II Δψ assay (
5 μM) and NAO (420 nM) were added to the cells according to the procedure of Example 1. Ionomycin (50 μM) in medium was also added to one set (n = 24) of samples, and medium alone was added to control set samples. Δ
The ψ-dropping agent CCCP (5 μM) was added to all samples after approximately 9 minutes. Fluorescence was measured using the FLIPR ^™ instrument during this experiment, as described above.

The results are shown in FIG. Ionomycin (“I”) resulted in a large proportion of Δψ loss, while carcinoma cells recovered relatively quickly about 6 minutes after addition of ionomycin. This recovery was different from that found in the cytoplasmic hybrid cell line used in the previous example and that found in the neuroblastoma SH-SY5Y cell line. This suggested that mitochondria in carcinoma cell lines take up TMRM more rapidly than mitochondria from other cell types, either generally, or at least after challenge to Δψ. FR of mitochondrial membrane potential
It is particularly noted that the different sensitivities to ionomycin detected in the ET assay, as demonstrated herein, can be used to distinguish cell types, which are different sensitivities to the ΔΨ plummet inducer: NCI-
Ionomycin concentration used herein for H460 cells (50 μM)
The (concentration at which these cells were recovered) was 10 times the ionomycin concentration at which SH-SY5Y cells were sensitive, as indicated by the loss of their mitochondrial membrane potential (Figure 12). As described above, FIG. 12 shows dequenching of NAO fluorescence (de) at higher ionomycin conditions using SH-SY5Y cells.
Quenching), which shows a greater drop in mitochondrial membrane potential at higher ionomycin concentrations, which affects the loss of TMRM from the mitochondrial fraction.

Example 9 Methods for Identifying Compounds for Treating Stroke Mechanisms of cell death from ischemia and reperfusion include both necrosis and delayed apoptosis, for both It is accompanied by mitochondrial dysfunction as a common precursor state. Numerous events following ischemia-induced loss of mitochondrial function include decreased mitochondrial energy metabolism, increased mitochondrial production of toxic reactive oxygen species (ROS) after reperfusion, and conditions under which energy production can be repaired. And the initiation of the active mitochondrial apoptotic cascade.

Following a neuronal ischemic event, mitochondrial ATP production ceases due to lack of oxygen. Glycolysis ATP production can continue under anoxic conditions, but glycolysis cannot meet neuronal energy requirements due to limited storage of glycolytic substrates in the brain. In addition, lactate accumulates in anoxic brain tissue and provides a measurable endpoint for biological assays. Due to the loss in aerobic capacity, tissue ATP levels drop to negligible levels shortly after cessation of oxygen flow to the brain.

[0241]   Without sufficient ATP, ATP-dependent ion transport is reduced and
Homeostasis loss occurs in the anoxic core of infarction
Resulting in osmotic lysis and necrosis of neurons. De-energization (de-ene)
is also an ATP-dependent transport process that sequesters glutamate.
Including loss of space. Ca²⁺And large influx of other ions
Occurs after activation of channels and ligand-gated ion channels (White
  et al. J. Neurosci. 15: 1318-1328, 1995.
Harrington et al. , Neuron 16: 219-228.
, 1996; Schinder et al. J. Neurosci. 16:
6125-6133, 1996). High levels of cytosolic Ca during reperfusion ²⁺ Directly activates mitochondrial calcium uptake during oxygen reintroduction
Inhibits the establishment of normal mitochondrial function. Excessive Ca in mitochondria ²⁺ Accumulation is the production of oxygen- and carbon-centered radicals in neurons
And can lead to inactivation of the mitochondrial electron transport chain (Dyk
ens, J.M. Neurochem. 63: 584-591, 1994; Reyn.
olds et al. Neurosci. 15: 3318-3327, 1
995; Dugan et al. J. Neurosci. 15: 6377-
6388, 1995, Bindokas et al. J. Neurosci
． 16: 1324-1336, 1996).

Another consequence of mitochondrial Ca ²⁺ uptake is the induction of transmembrane permeability (MPT), which consumes ΔΨ _m and solutes <1,500 daltons across the internal mitochondrial membrane. Specific-specific voltage-sensitive pore opening that allows equilibration (Review, Zoratti et al., Biochim. B).
iophys. Acta 1241: 139-176, 1995; Bernar.
di et al. J. Bioenerg. Biomemb. 26: 509-
517, 1994). The high ΔΨ _m normally produced by the electron transport chain in the absence of high Ca ²⁺ or free radical-induced damage is a strong interferer to MPT pore formation. Factors that mitigate MPT and ΔΨ _m plunges (eg, Bcl-2 and cyclosporin A) likewise alleviate glutamate excitotoxicity both in vitro and in vivo (Hoyt et al., B.
r. J. Pharmacol. 122: 803-808, 1997; Niemn.
inen et al. , Neurosci. 75: 993-997, 1996.
Ankarcrona et al. , FEBS Lett. 394: 321
-324, 1996; Uchino et al. , Acta Physiol
． Scand. 155: 469-471, 1995; Li et al. , Br
ain Res. 753: 133-140, 1997).

Failure of the cellular Ca ²⁺ efflux mechanism and activation of phospholipases and proteases appear to be a late stage event after ischemia and can result in widespread injury to membranes and proteins. Cells exposed to less severe stress can initiate the apoptotic cascade. In this case, the mitochondria can be reversibly damaged and release sufficient levels of apoptotic factors to induce death, but retain the remaining ability to produce ATP (MacManu).
s et al. J. Cerebral Blood Flow Metab
． 17: 815-832, 1997). Therefore, healthy mitochondria play a bifunctional role in preventing neuronal survival in ischemia / reperfusion injury:
1) By supplying ATP, mitochondria provide the driving force for reuptake of glutamate from the synaptic cleft, and the ATP-dependent maintenance of normal membrane potential that further interferes with the opening of voltage-sensitive ion channels, and 2) The intact mitochondria resist the release of factors that can direct neurons to the underlying apoptotic pathway. Therefore, maintaining mitochondrial integrity during ischemia / reperfusion and thereby protecting against subsequent waves of excitotoxicity has utility for preventing stroke-related neuronal injury, Allows the identification of novel neuronal cell protective factors.

Primary Screening Assay The measurement of ΔΨ _m provides a comprehensive indicator of mitochondrial function and integrity. Thus, the primary screening assay in seizure drug discovery utilizes an ET-based assay of ΔΨ in whole cells in a high throughput format. Factors and methods that maintain mitochondrial integrity during transient ischemia, and post-excitotoxic waves are effective neuroprotective factors that have utility in limiting stroke-related neuronal injury. Expected to be. To develop a therapeutic strategy to limit neuronal death by preventing mitochondrial dysfunction in the non-necrotic region of the infarct, considering the limits of therapeutic window for obstructing necrotic death in the center of the infarct Is especially desirable. To this end, compounds are screened for their effect on ΔΨ under control and Ca ²⁺ overflow conditions.

After stroke, most of the damage to neurons in the border zone is brought about by excitotoxicity induced by glutamate released during cell lysis in infarct lesions. To more closely mimic in vivo biochemical and cellular events, primary screening assays were performed in cells containing one or more types of glutamate receptors (for a review, see Gasis et al., Curr.
． Opin. Neurobiol. 1: 20-26, 1991; Westbro
ok, Curr. Opin. Neurobiol. 4: 337-346, 199.
4; and Lynch et al. Curr. Opin. Neurobio
l. 7: 510-516, 1994).

The glutamate receptor is an ion channel type glutamate receptor (iG
luR) and metabotropic receptors (mGluR). iGluR is a glutamate-gated cation channel. This cation channel is
It is classified into subclasses of NMDA receptor, AMPA receptor and kainate receptor. NMDA receptors include, for example, NMDAR1 / 2A, NM
A heteromeric complex containing DAR1 / 2B, NMDAR1 / 2C and NMDAR1 / 2D. AMPA receptors include, for example, GluRl, GluR2
, GluR3 and GluR4 are homomeric complexes. The kainate receptor can be either a homomeric or heteromeric complex of GluR5, GluR6, GluR7, KA-1 and KA-2. mG
luR is a 7-transmembrane G protein-coupled receptor, which also falls into a subclass. Some mGluRs are phospholipase C-bound mGluRs that increase cytosolic calcium: mGluRl and m
Includes GluR5. Other mGluRs are adenyl cyclase-linked mGluRs that reduce cytosolic cAMP; these include mGluR2, mGluR3,
Included are mGluR4, mGluR7, and mGluR8.

One example of cells containing one or more types of glutamate receptors used in primary screens are primary cortical neurons that express the endogenous NMDA receptor. In these cells, provision of extracellular glutamate increases intracellular calcium levels (Stout et al., Nat. Neurosci.
1: 366-373, 1998). Subsequent to glutamate addition, changes in ΔΨ are measured using an ΔET ET-based assay. Mitochondrial deficient cytoplasmic hybrid cells with suppressed ΔΨ (Cassarino et al.,
Biochem. Biophys. Res. Commun. 248: 168-1
73, 1998) also provides a broader response to factors and / or conditions that have been utilized in addition to primary neuronal cell cultures and tested for their ability to consume or plummet ΔΨ.

Other examples of cells containing one or more types of glutamate receptors used in the primary screen include cells that have been genetically engineered to express or overexpress one or more glutamate receptors. Many mammalian cell lines
It was engineered to stably express the glutamate receptor in culture (for a review, Varney et al., Methods. Mol. Biol.
128: 43-59, 1999). Non-limiting examples of glutamate receptors cloned and expressed in mammalian cells include NMDRA
1A / 2A and NMDAR1A / 2B (Varney et al., JP
armacol. Exp. Ther. 279: 367-378, 1996);
NMDAR2C, isoforms 1, 2, 3 and 4 (Dagget et a
l. J. Neurochem. 71: 1953-1968, 1998); Gl
uR3 (Varney et al., J. Pharmacol. Exp. Th.
er. 285: 358-370, 1998); and GluR1b and Gl.
uR5a (Lin et al., Neuropharmacology 36
: 917-931, 1997).

[0249]   (Secondary screening assay)   High [Ca in primary assay²⁺ _i] Resulting from ΔΨ_mExtended
Compounds that prevent plunge were further evaluated in a secondary assay, which included RO
S production, cytochrome c release and caspase-3 activity as indicators of apoptosis
Includes measurements of denaturation and cell viability. In this way, FRET ΔΨ_mAsse
The “hit” identified in b) was further verified, and the compound was _m The mechanism that influences can be better defined. Reasons for these assays
The rationale is that mitochondrial integrity under conditions of excitotoxicity or oxidative stress.
A compound capable of maintaining sex is correspondingly released of apoptogen.
And borderline neutrophils that may reduce apoptotic and postapoptotic death after transient ischemia.
Based on evidence suggesting that it may be possible to rescue Ron. The following assay
Time-pending US patent application serial number 09 / 299,044 (April 23, 1999)
Application) for more details.

Assay for Inhibition of Reactive Oxygen Species Production Using Dichlorofluorescein Diacetate: According to this assay, the mitochondrial protective agent is
The ability to inhibit the production of OS intracellularly can be compared to the antioxidant activity of this protectant in a cell-free environment. The production of ROS is, for example, for purposes of illustration and not limitation, a sensitive indicator of the presence of oxidizing species, 2 ', 7'-dichlorodihydrofluorescin diacetate ("dichlorofluororescin diacet").
ate ”or DCFC). The non-fluorescent DCFC is
Upon oxidation, it is converted to a fluorophore that can be quantified fluorescently. The cell membrane is also DC
Although permeable to FC, DCFC's charged acetate groups are removed by intracellular esterase activity, which weakens the ability of this indicator to diffuse from the cell.

In the cell-based aspect of the DCFC assay for inhibition of ROS production, cultured cells can be preloaded with an appropriate amount of DCFC and then contacted with a mitochondrial protective agent. After a suitable interval, the production of free radicals in cultured cells can be induced by contacting them with iron (III) / ascorbate,
And the relative mean DCFC fluorescence can be monitored as a function of time.

In the cell-free aspect of DCFC for inhibition of production of ROS, the mitochondrial protective agent is capable of producing iron / It can be tested for its ability to directly inhibit ascorbate-induced DCFC oxidation.

A comparison of mitochondrial protective agent properties in cell-based and cell-free aspects of the DCFC assay determines whether inhibition of ROS production by mitochondrial protective agents proceeds stoichiometrically or catalytically. Can be enabled. Without wishing to be bound by theory, a mitochondrial protective agent that scavenges free radicals stoichiometrically (eg, on a 1: 1 molecular basis) may not mean a preferred agent. This is because such agents appear to require high intracellular concentrations to be effective in vivo. On the other hand, may catalytically acting mitochondrial protective agents regulate the production of oxygen radicals at their source,
Alternatively, the protectant itself may block ROS production without modification, or it may alter the reactivity of ROS by unknown mechanisms. Such mitochondrial protectors can be "recycled", so mitochondrial protectors can inhibit ROS below stoichiometric concentrations. The determination of this type of catalytic inhibition of ROS production by mitochondrial protective agents in cells indicates the interaction of this agent with one or more cellular components that synergize with this agent to diminish or prevent ROS production. obtain. Mitochondrial protective agents having such catalytic inhibitory properties may be preferred agents for use according to the methods of the invention.

Mitochondrial protective agents useful according to the invention may inhibit ROS production, as quantified by fluorescent assays or other assays based on similar principles.
Those skilled in the art are familiar with variations and modifications that can be made to assays such as those described herein without departing from the essence of this method of determining the efficacy of mitochondrial protective agents, and such Variations and modifications are within the scope of this disclosure.

(2,4-Dimethylaminostyryl-N-methylpyridinium (DASPMI
Assay for changes in mitochondrial permeability (MPT)): According to this assay, one skilled in the art determines the ability of the mitochondrial protective agent of the invention to inhibit the loss of mitochondrial membrane potential associated with mitochondrial dysfunction. You can As noted above, maintenance of mitochondrial membrane potential can be convinced as a result of mitochondrial dysfunction. This loss of membrane potential or changes in mitochondrial permeability (MPT) can be measured quantitatively using the mitochondrial-selective fluorescent probe 2-, 4-dimethylaminostyryl-N-methylpyridinium (DASPMI).

Upon introduction into cell culture, DASPMI accumulates in mitochondria in a manner that is dependent on and proportional to the mitochondrial membrane potential. When mitochondrial function is disrupted in a manner that matches the membrane potential, the fluorescent indicator compound leaks out of the membrane-bound organelle with a loss of detectable fluorescence. DASP
The fluorescence potential of mitochondrial decay associated with MI fluorescence was measured fluorometrically,
That is, a quantitative measure of MPT loss is provided. Mitochondrial dysfunction
Mitochondrial protective agents that slow the rate of loss of DASPMI fluorescence, as they can be the result of reactive free radicals such as ROS, can be effective agents for treating disease-related mitochondria according to the methods of the invention. .

Assay for apoptosis in cells treated with mitochondrial protective agent:
As mentioned above, mitochondrial dysfunction may be an inducing signal for cellular apoptosis. Following the assays in this section, one of skill in the art can determine the ability of the mitochondrial protective agents of the invention to inhibit apoptosis or delay expression. Mitochondrial dysfunction may be present in cells known or suspected to be from a subject with a mitochondrial-related disorder, or mitochondrial dysfunction may be due to a variety of physical (eg, UV irradiation),
By one or more of physiological and biochemical stimuli, familiar to those skilled in the art,
It can be induced in cultured normal or diseased cells.

Apoptosis and / or biochemical processes associated with apoptosis may also be associated with one or more "apoptogens" (ie, when contacted with or recovered from cells or isolated mitochondria). , Factors that induce apoptosis and / or related processes). Such apoptogens are for illustrative purposes and include, but are not limited to, the following: (1) Apoptogens added to cells having specific receptors therefor, such as tumor necrosis factor (TNF), FAsL. , Glutamate and NMDA; (2) growth factors recovered from cells having specific receptors for such factors, such factors include, for example, IL-3 or corticosterone; and added to cells Apoptogens that can be generated but do not require specific receptors, including (3) Herbimycin A (Ma
ncini et al. Cell. Biol. 138: 449-469, 1997).
, (4) Paraquat (Cosantini et al., Toxicology 99:
1-2, 1995); (5) ethylene glycol (http: //www.ul.
aval. ca / vrr / rech / Proj / 532866. html); (
6) Protein kinase inhibitors, for example: staurosporine, calphostin C, d-erythro-sphingosine derivatives, chelerythrine chloride, genistein, 1- (5-isoquinolinesulfonyl) -2-methylpiperazine, KN.
-93, quercitin, N- [2-((p-bromocinnamyl) amino) ethyl] -5-5-isoquinolinesulfonamide and caffeic acid phenethyl ester; and (7) ionophores, for example: ionomycin and valinomycin. (8) MAP kinase inducer, for example,
(9) cell cycle blockers, such as anisomycin and anandamine;
The following: aphidicolin, colcemid, 5-fluorouracil, and homoharringtonine etc .; (10) acetylcholinesterase inhibitors such as berberine; (11) anti-estrogens such as tamoxifen; For example, tert-butyl peroxide and hydrogen peroxide; (13) free radicals such as nitric oxide; (14) inorganic metal ions such as cadmium; (15) DN.
A synthesis inhibitors such as actinomycin D, bleomycin sulfate, hydroxyurea, methotrexate, mitomycin C, camptothecin, daunorubicin and the like, and intercalators such as doxorubicin and the like; (16
) Protein synthesis inhibitors such as cyclohexamide, puromycin and rapamycin; (17) Factors affecting microtubule formation or stability such as vinblastine, vincristine, colchicine, 4-hydroxyphenylretinamide and paclitaxel; 18) Factors that increase intracellular calcium levels by releasing intracellular calcium from intracellular reservoirs, such as thapsigargin (Thastrup et al., Proc. Natl. Acad. Sci. US).
． A. 87: 2466-2470, 1990), thapsigarginine (thaps)
igargicin) (Santarius et al., Toxicon 25: 389.
~ 399, 1987), etc., and excitatory amino acids and their derivatives, such as kainic acid, N-methyl-D-aspartic acid (NMDA), N-acetylaspartyl glutamate (NAAG, glutamate derivatives), 2-amino-.
3- (3-hydroxy-5-methylrisoxazol-4-yl) propionic acid (
AMPA) and 2-amino-3- (3-hydroxy-5-phenylisoxazol-4-yl) propionic acid (APPA, AMPA derivatives) and the like; and factors added to isolated mitochondria, such as ( 19) MPT inducer,
For example, Bax protein (Jurgenmeier et al., Proc. Natl. A.
cad. Sci. U. S. A. 95: 4997-5002, 1998); and (20) calcium phosphate and inorganic phosphate (Kroemer et al. Ann.R.
ev. Physiol. 60: 619-642, 1998).

In one aspect of the apoptosis assay, cells suspected of undergoing apoptosis can be tested for morphological, permeability or other alterations indicative of apoptotic status. For example, and for purposes of illustration and not limitation, apoptosis in many cell types may have altered morphological appearance (eg, plasma membrane vesicle formation, cell shape change, loss of substrate adhesion properties or using light microscopy). And other morphological changes readily detectable by one of skill in the art. In another example, cells undergoing apoptosis may exhibit chromosomal fragmentation and degradation. This may be evident by microscopy and / or through the use of DNA-specific or chromatin-specific dyes (known in the art, including fluorescent dyes). Such cells may also exhibit altered membrane permeability properties. This may be readily detectable through the use of vital dyes (eg propidium iodide, trypan blue) or detection of lactate dehydrogenase leakage into the extracellular environment. Damage to DNA can also be assayed using electrophoretic techniques (eg, Morris et al., BioT.
techniques 26: 282-289, 1999). These and other means for detecting apoptotic cells by morphological, permeability and associated changes will be apparent to those of skill in the art.

In another aspect of the apoptosis assay, translocation of plasma membrane phosphatidylserine (PS) from the interior to the outer leaflets of the plasma membrane is quantified by measuring outer leaflet binding by the PS-specific protein annexin. (Martin
Et al., J. Exp. Med. 182: 1545-1556, 1995; Fadok
Et al., J. Immunol. 148: 2207-2216, 1992). In a preferred manner, the externalization of plasma membrane PS is achieved by annexin-fluorescein isothiocyanate conjugate (annexin-FITC,
Oncogene Research Products, Cambridge
, MA) using labeled annexin derivatives in 96-well plates.

In another aspect of the apoptosis assay, quantification of the mitochondrial protein cytochrome c leaked out of mitochondria in apoptotic cells can provide an easily determinable apoptotic index (Liu et al. Cell 86:
147-157, 1996). Such quantification of cytochrome c can be performed spectrophotometrically, immunochemically, or by other well-established methods for detecting the presence of a particular protein. Release of cytochrome c from mitochondria in cells challenged with apoptotic stimuli, such as ionomycin, a well-known calcium ionophore, can be followed by various immunological methods. Flight mass spectrometry combined with affinity capture (MALDI
The matrix-assisted laser desorption ionization time of -TOF) is particularly suitable for such analysis. This is because apocytochrome c and holocytochrome c can be distinguished based on their unique molecular weights. For example, the SELDI system (Ciphergen, Palo Alto, USA) can be used to follow inhibition of mitochondrial protective release of cytochrome c from mitochondria in ionomycin treated cells. In this approach, a cytochrome c-specific antibody immobilized on a solid support is used to capture the released cytochrome c present in the soluble cell extract. The captured proteins are then encased in a matrix of energy absorbing molecules (EAM) and detached from the solid support surface using pulsed laser excitation. The molecular weight of this protein is determined by its time of flight to the detector of the SELDI mass spectrometer.

In another aspect of the apoptosis assay, induction of specific protease activity in a family of apoptosis-activating proteases known as caspases (
Thornberry and Lazebnik, Science 281:
1312 to 1316, 1998) are measured, for example, by determining caspase-mediated cleavage of specifically recognized protein substrates. These substrates may include: for example, poly- (ADP-ribose) polymerase (PARP).
Or other naturally-occurring or synthetic peptides and proteins (cleaved by caspases known in the art) (eg, Ellerby et al., JN).
eurosci. 17: 6165-5178, 1997). Labeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (where "Z"
Indicates a banzoylcarbonyl moiety, and AFC indicates 7-amino-4-trifluoromethylcoumarin) (Kluck et al., 1997 Science.
275: 1132-1136, 1997; Nicholson et al., Nature.
376: 37-43, 1995) is one such substrate. Another labeled synthetic peptide substrate for caspase-3 is the protease recognition site /
It is composed of two fluorescent proteins linked to each other via a peptide linker containing a cleavage site (Xu et al., Nucleic Acids Res. 26: 203.
4-2035, 1998). Other substrates include U1-70kDA and DNA
-Nuclear proteins such as PKcs (Rosen and Cascio
la-Rosen, J .; Cell. Biochem. 64: 50-454,19
97; Cohen, Biochem. J. 326: 1-6, 1997).

In another aspect of the apoptosis assay, the ratio of dead to dead cells or the ratio of dead cells in the population of cells exposed to apoptogen is measured as a measure of the final outcome of apoptosis. Viable cells can be distinguished from dead cells using any of a number of techniques known to those of skill in the art. As a non-limiting example, biogenic dyes such as propidium iodide or trypan blue can be used to measure the percentage of dead cells in a population of cells treated with apoptogen and a compound according to the invention.

One of skill in the art will recognize that other suitable techniques may exist for quantifying apoptosis,
And it is readily appreciated that such techniques for measuring the effect of mitochondrial protective agents on the induction and kinetics of apoptosis are within the scope of the assays disclosed herein.

(Assay of Electron Transfer System (ETC) Activity in Isolated Mitochondria)
: As noted above, mitochondrial-related disorders can be characterized by impaired mitochondrial respiratory activity. This disorder can be a direct or indirect result of elevated levels of reactive free radicals such as ROS. Accordingly, mitochondrial protective agents for use in the methods provided by the invention may restore or prevent further reduction of ETC activity in mitochondria of individuals with mitochondrial-related disorders. Mitochondrial ETC Complex I, II, III, IV
And an assay method for monitoring the enzymatic activity of ATP synthetase,
As well as assay methods for monitoring oxygen consumption by mitochondria are well known in the art. (For example, Parker et al., Neurology.
44: 1090-1096, 1994; Miller et al. Neuroche
m. 67: 1897-1907 1996). It is within the scope of the methods provided by the present invention to identify mitochondrial protective agents using such assays of mitochondrial function.

In addition, mitochondrial function determines the oxidation state of mitochondrial cytochrome c by 5
It can be monitored by measuring at 40 nm. As mentioned above, oxidative damage that can be elevated in mitochondrial-related diseases can include damage to mitochondrial components, such that the cytochrome c oxidation state can by itself or other parameters of mitochondrial function (mitochondrial oxygen. Combined with, but not limited to, consumption) and may be an indicator of reactive free radicals for mitochondrial components. Accordingly, the present invention provides various assays designed to test the inhibition of such oxidative damage by mitochondrial protective agents. Various forms of such assays can be considered as understood by those of skill in the art and are not intended to be limited by the disclosure herein, including the disclosure in the Examples, eg, by way of example and limitation thereto. For the purposes not being performed, complex IV activity can be determined using commercially available cytochrome c which has been completely reduced by exposure to excess ascorbate. Cytochrome c oxidation can then be monitored spectrophotometrically at 540 nm using a stirred cuvette where the ambient oxygen of the buffer has been replaced with argon. Oxygen reduction in the cuvette can be monitored simultaneously using micro oxygen electrodes familiar to those skilled in the art, where such electrodes are used in a manner that maintains the argon atmosphere of the sample (e.g., sealed rubber). Through the stopper) and into the cuvette. The reaction may be initiated by the addition of cell homogenates or preferably isolated mitochondrial preparations via injection through a rubber stopper. This assay, or other assays based on similar principles, may allow mitochondrial respiratory activity to be correlated with structural features of one or more mitochondrial components. In the assays described herein, for example, the lack of complex IV activity can be correlated with the enzyme recognition site.

Tertiary Screening Assay Compounds possessing the desired activity profile in a second in vitro assay were tested for transient focal ischemia in a rodent middle cerebral artery occlusion (MCAO) model, which
It has been reported to produce ischemia similar to MCAO branch occlusion in humans (L
inga et al., Stroke 1: 84-91)). First, test compounds are administered by continuous intravenous infusion before and during the ischemia / reperfusion period to ensure a good opportunity for successful experiments. Once efficacy is established, experiments are conducted using single and multiple drug dosing regimens to assess efficacy as post-treatment. The potency of the test compound is
It is assessed directly by measuring the reduction of neuronal loss in the infarcted brain region using techniques such as magnetic resonance imaging. Other additional endpoints are then measured, including decreased lactic acid production in the brain, decreased DNA, protein and lipid oxidation products as a result of conversion from aerobic to anaerobic metabolism after hypoxia.

Example 10 ET-Based Assays for Monitoring Subcellular Fraction Fusions Energy transfer-based assays are used to monitor subcellular fraction (eg, organelle) fusions. obtain. For example, mitochondria undergo changes including division and fusion, and the latter process involves apparently coordinated rearrangements of internal elements (ie, inner membrane, crystals, etc.) (for review, see Ber.
Eiter-Hahn and Voth, Microscopy Research
h and Technique 27: 198-219, 1994). Such changes are believed to be important for various developmental processes. Various organisms (yeast (eg C. cervisiae), insects (eg D.
． In melanogaster, invertebrates (eg, C. elegans) and mammals (eg, H. sapiens), mitochondrial fusion results in “mitofusin” (Hales et al., Cell 90).
: 121-129, 1997; Hermann et al., J. Am. Cell. Biol. 1
43: 359-373, 1998; and published PCT patent application WO98 /
55618) and is mediated by the GTPase protein. Fuzzy onion (fzo) gene, which encodes mitofusin in D. melanogaster
Mutations in sperm impair spermatogenesis and render male insects sterile (Hale
s et al., Cell 90: 121-129, 1997).

Thus, in certain embodiments, the invention modifies mitochondrial fusion by assaying a mitochondrial fusion event in the presence or absence of a candidate agent (ie, in a statistically significant manner). Methods for identifying agents that increase or decrease) are provided. Such an agent comprises contacting a first sample containing one or more mitochondria with an ET donor molecule with a second sample containing one or more mitochondria with an ET acceptor molecule, mitochondrial fusion. Contacting the first and second samples with each other in the presence and absence of the candidate agent under conditions and for a time sufficient to allow the excited E
Exciting an ET donor to produce a T donor molecule, ET donor to ET
It is identified by detecting the signal produced by energy transfer to the receptor, and comparing the signal produced in the absence of the candidate agent to the signal produced in the presence of the candidate agent.

The present invention relates to methods for identifying agents that alter mitochondrial fusion, in certain preferred embodiments thereof, neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and The ET donor and ET acceptor are each independently located at the same sub-mitochondrial site or an acceptably adjacent sub-mitochondrial site as provided herein. Typically, based on the techniques provided herein, one of skill in the art will know when a candidate agent alters a mitochondrial fusion, eg, E produced in the absence of the agent.
It can be readily determined by detecting a statistically significant change in the ET signal generated in the presence of the agent relative to the T signal. As mentioned above, conditions that allow mitochondrial fusion events are known in the art, and as a result, how can one of skill in the art perform immediate assay methods without undue experimentation. It can be easily determined whether the appropriate conditions are appropriate. For purposes of illustration and not limitation, such conditions may include conditions that allow the fusion of isolated mitochondria, which refers to mitochondria removed from the environment in which the mitochondria naturally occur; Such conditions also include conditions that allow the mitochondria of at least one sample population to fuse intracellularly.

Works by selectively inhibiting mitofsin activity in unwanted insects or eukaryotic parasites, but with minimal effect on the desired insect or plant or mitofsin in a mammalian host, including humans It is desirable to develop new antibiotics or pesticides that have no or no effect. It is also desirable to identify and characterize agents that stimulate or inhibit intracellular mitochondrial fusion events for the treatment of human disease. The present invention can be used to achieve these objectives in the following manner.

Generally, a first group of mitochondria is preincubated with a donor compound and a second group of mitochondria is incubated with an appropriate acceptor compound. Simultaneous incubation of the first and second groups of mitochondria results in the fusion of individual mitochondria from each set, where the donor and acceptor compounds are in close proximity to each other and are of interest. achieve. Thus, mitochondrial fusion results in energy transfer that can be measured according to the present disclosure. Also, when agents that stimulate or inhibit mitochondrial fusion are added to these reactants, the degree of energy transfer and / or the rate of energy transfer is increased or decreased, respectively. Therefore, candidate agents that affect the activity or expression level of mitofusin protein are screened for and characterized via an ET-based assay.

Example 11 ET-Based Assay for Monitoring Drug Localization to Specific Subcellular Sites Organelle or intact cells isolated using an energy transfer-based assay. The influx or efflux of drugs into specific subcellular fractions within can be monitored; in the latter case, such assays can be used to assess the pharmacokinetic properties of candidate therapeutic agents. For example, agents containing tetramethylrhodamine (TMR) or related moieties have been described. For example, an oligonucleotide labeled at the 5 ′ end with TMR is a Genomyx Corp (Foster City, CA).
) And linked to a rhodamine or dichlororhodamine moiety are available from Perkin-Elmer Corp. (No
rwalk, CT). General methods for preparing conjugates containing NAO-based moieties or JC-1 based moieties are described in published PCT patent application WO 98/17826. Mitochondrial uptake of such agents can be assessed using the present invention as follows.

Uptake of drugs containing tetramethylrhodamine (TMR) or related moieties into mitochondria is associated with donor compounds (eg NAO, MitoTracker (
It can be monitored by preincubating mitochondria or cells containing mitochondria for a period of time with the registered trademark) Green FM or MitoFluor ^™ Green), after which the TMR binding agent of interest is added. When the drug is taken up by mitochondria, its TMR or TMR-like portion acts as an acceptor for the energy emitted from the donor compound. Thus, uptake of this drug will reduce release from the donor,
Or it can be understood as a function of either increasing the release from the TMR or TMR-like moiety.

Similarly, mitochondrial uptake of agents containing NAO or TMR-like moieties can be achieved by preincubating mitochondria or cells containing mitochondria with a receptor compound (eg, TMRM, TMRE or rhodamine 123) for a period of time, followed by Of NAO binding agent can be monitored. When this agent is taken up by mitochondria, its NAO or NAO-like portion acts as a donor to the energy released from the acceptor. Thus, uptake of this drug can be understood as a function of either increasing release from the receptor compound or decreasing release from the NAO or NAO-like moieties. Uptake of drugs containing JC-1 based moieties is
It is monitored in a similar fashion, except that appropriate donor or acceptor compounds for JC-1 and mitochondria (see Tables 2 and 3) are used.

Example 12 FRET-Based Assay for ΔΨ in Permeabilized Cells In the following experiments, a ΔRET FRET-based assay using NAO and TMR was performed using other permeabilizing agents. However, essentially as described in Examples 5-7, except that the cells used in the experiments were typically permeabilized (unless otherwise indicated) by treatment with digitonin. Carried out. A related exception is that it is not necessary to add an ionophore (eg, ionomycin) to promote calcium entry into the cell because it has permeabilized the cell. Instead, calcium was added to the medium in the absence of an ionophore and allowed free access to permeabilized cells. Control experiments were carried out in the presence or absence of ionomycin, which ensures that calcium ions are free to enter permeabilized cells at least to the same extent as ionomycin-treated cells. It was demonstrated that the same response to different concentrations of calcium was observed in permeabilized cells.

Another notable difference between the assay protocol described in this example and the assay protocol described above is that cells are treated simultaneously with ET donor compound and ET acceptor as in Examples 5-7. This means not contacting both body compounds. Instead, cells were first contacted with NAO alone (0.04 μM; 5 minutes) and rinsed three times at 37 ° C. in HBSS buffer (although many other buffers are suitable for these rinses). , And then a plate reader (ie,
An instrument capable of reading the signal produced due to energy transfer in the assay, preferably an automated or semi-automated instrument (eg FLI as described above).
It is a PR ^TM device)).

Once in the plate reader, the signal due to NAO was monitored for about 1 minute before adding TMR as the second member of the ET pair. As described above, and according to a non-limiting theory, TMR is used as the NAO-derived fluorescent signal in F
Accumulates in mitochondria via RET as a function of ΔΨ and quench. As ΔΨ changes in response to different compounds or conditions, the concentration of TMR in mitochondria changes in a corresponding manner, as reflected by the change in signal corresponding to NAO quenching.

[0279] Typically, the average of several readings during this interval was taken for analysis and labeled "Q" in Figure 16. Quenching of NAO by TMR was monitored in real time to ensure equilibrium was reached prior to addition of test compound and / or other agents. This steady state is indicated as "R" in FIG. Cells were washed and all reagents were added to 20 mM HE.
Hanks Balanced Salt Solutio with PES buffer
n (HBSS). The test compound is then added and allowed to equilibrate for at least 5 minutes; the average of several readings corresponding to later intervals is labeled "S" in FIG. At the final stage of the assay (denoted as "T" in Figure 16), ΔΨ was plunged by adding Ca ²⁺ directly to permeabilized cells. This step differs from the protocol of the previous example, where Ca ²⁺ mediated changes in ΔΨ were facilitated by ionophores (eg, ionomycin) that facilitated the entry of high concentrations of Ca ²⁺ from the extracellular environment into the cytosol. ) Induced in intact (non-permeabilized) cells.

In the protocol used in this example, TMR was present throughout the assay after TMR was added to the assay. This minimized the potential leakage of TMR from the mitochondria after washing and therefore stabilized the baseline readings; it removed TMR prior to placement in the plate reader. The cells were washed and were not characteristic of the protocol described in the previous example.

Another modification of the assay in this example involved data processing. The data were analyzed by dividing the signal collected after Ca ²⁺ addition (difference TS) by the signal initially quenched by TMR (difference QR). These mathematical treatments were performed according to the ratio (ie, “ΔΨ-variance value (Dissipat)
ing Value) "and" Efficacy Index "
) Produced a different ratio. For example, using the data shown in FIGS. 16 and 17, according to the following equation, “recovery of initial quenching (Ratio of of
Recovery of Initial Quenching ”(hereinafter“ RPIQ ”).

RPIQ = [T−S] / [Q−R] An agent that inhibits or blocks a ΔΨ plunge is an RRIQ below the RRIQ of a control sample exposed to Ca ²⁺ but not treated with such an agent. Have. Conversely, compounds that induce or enhance a ΔΨ drop are not treated with the untreated control R
It has a larger RRIQ than the RIQ.

It is also possible to evaluate the direct effect of the compounds on ΔΨ by examining the values corresponding to the difference between R and S (ie RS). Hyperpolarized drug (
For example, oligomycin) increases TMR uptake and therefore NAO quenching, which results in a positive value for RS. In contrast, compounds that directly consume ΔΨ produce negative values for RS.

Validation studies showed that Ca ²⁺ showed a dose-dependent response, where high Ca ²⁺ levels caused ΔΨ to plummet to initial levels with only minimal recovery of ΔΨ. It was However, intermediate Ca ²⁺ levels allowed some recovery of ΔΨ. ΔΨ is frequently lost when mitochondria undergo a second permeability change. That this was indeed the second change was supported by the observation that such changes were inhibited by cyclosporin A and bongcrekinic acid. In many cases, the data from such situations show that the area under the curve (AUC) for a constant duration after Ca ²⁺ addition, rather than using the average response for the specified intervals.
Was best analyzed by summing.

In addition to the ability to monitor a second ΔΨ plunge due to permeability changes, the protocol used in this example also identified agents that affect mitochondrial Ca ²⁺ uptake (eg, RU-360). To enable. In this case, the initial depolarization upon Ca ²⁺ addition was reduced compared to untreated controls. Analysis of C
Achieved by comparison with the maximum peak height immediately after a ²⁺ addition.

As shown in FIG. 17, permeabilized cells treated with calcium alone (ie, without the presence of an ionophore) undergo a concentration-dependent response to calcium ions, with ΔΨ in terms of both extent and time course of response. Plunge drug (ΔΨ-colla
The plunge seen in cells treated with CCCP, which is a paging agent), leads to a plunge in ΔΨ with approximately 100 μM Ca ²⁺ . 16 and 17
The data shown in Figure 2 was generated using SH-SY5Y cells permeabilized with digitonin (0.008% v / v) obtained from Sigma (St. Louis, MO). Cells were treated with 125 mM KCl, 2 mM K ₂ HPO ₄ , 5 mM HEPES.
It was grown in a medium containing 4 mM MgCl ₂ , 1 mM maleic acid, 1 mM succinic acid, 1 mM glutamic acid, 1 μM EGTA, pH 7.0.

In FIG. 18, the RRIQ derived from the data shown in FIGS. 16 and 17 is plotted as a function of calcium ion (Ca ²⁺ ) concentration (log M). This concentration-response curve (hereinafter "CRC") is 56 for Ca ²⁺ in permeabilized cells.
． An EC ₅₀ of 5 μM is produced.

The data shown in the third embodiment is intact (that is, is not transparentized).
In various cells, ruthenium red, an inhibitor of mitochondrial calcium uniporter, regulates [ionomycin + Ca ²⁺ ] and induces a -ΔΨ drop (
For example, FIG. 8) is demonstrated. In these experiments (Example 12), as a control, an ionophore (ionomycin) was used to facilitate Ca ²⁺ entry into the cytosol of cells. FIG. 19: RU-360 (concentration 0-25 nM) (inhibitor of calcium uniporter, which is more specific in digitonin-permeabilized cells treated with Ca ²⁺ than ruthenium red). Shows the CRC of. Results are permeabilized and resulting an EC ₅₀ of 11nM for RU-360 in Ca ²⁺ treated cells. Unlike the results shown in Figures 18 and 20 (where RRIQ is plotted as a function of concentration of candidate or control agent), the response in Figure 19 is for each concentration of RU-360 rather than RRIQ value. It was summarized by calculating the area under the curve (see Example 4 and Table 6).

The data presented in Example 3 also shows that in intact cells cyclosporine A
It is shown that (hereinafter “CsA”) regulated [ionomycin + Ca ²⁺ ] and induced a sharp drop in ΔΨ at a concentration of −10 μM (eg, FIG. 9). In digitonin permeabilized cells treated with CsA, CsA relaxed Ca ²⁺ and induced a ΔΨ plunge with an EC ₅₀ of −0.31 μM (FIG. 20). The results shown in Figure 20 demonstrate the following important advantage of using permeabilized cells instead of intact cells for this type of assay: CsA shows a relatively mild ability to enter intact cells. (Thus reducing the apparent intracellular activity of CsA in intact cells) but is free to enter permeabilized cells.

The results presented in this example demonstrate various useful embodiments of the assay of the present invention utilizing permeabilized cells. In accordance with one such embodiment, a range of distinct permeabilization conditions is used to impair the cell's plasma membrane permselectivity, but to reduce organelle membranes (eg, mitochondria and / or chloroplast perimeter membranes). Or both) determine the conditions under which their selective permeability is maintained.

Permeabilization conditions, such as the conditions described in the previous section, may increase the activity of a particular organelle component from the group of candidate agents to achieve one or more of a variety of purposes that may include: Desirable in certain screening assays designed to select or identify individual active compounds that affect: (1) "false negative" (which
Due to all or part of the cells' moderate or limited ability to cross the plasma membrane, shows no activity in assays using intact (non-permeabilized) cells, ie, Avoiding failure to detect activity of candidate organelle-affecting agents; (2) preferentially preferring active compounds that directly or indirectly cause selective permeability of one or more membranes around the organelle. Or selectively, identifying or (3) allowing simultaneous contact of mitochondria with two or more agents, wherein each such agent is associated with the activity of a particular organelle component. Each of these agents affects and otherwise requires specific means by which it can enter the cytosol. In the case of objective (3), the objective of this assay may be to select or identify from a group of candidate agents, active compounds that are antagonists or agonists of agents that affect the activity of specific organelles and organelle components. . In the latter situation, permeabilization of the cells allows contacting the organelle with (i) an agent that affects the activity of a particular organelle component and (ii) one or more candidate agents. This treatment has the desirable feature of contacting the organelle with both (i) and (ii) with simultaneous and minimal manipulation of the cells used in this assay; such a feature has high throughput ( HTS
) Particularly useful in assays. As an example of one of the desirable aspects in achieving objective (3) in such an assay, the results presented herein indicate that mitochondrial Ca ²⁺ in permeabilized cells (which is otherwise And requires the presence of an ionophore, such as ionomycin, to facilitate Ca ²⁺ entry into the cytosol and boncrekinic acid (ANT (adenine nucleotide translocator) (a protein localized to the mitochondrial membrane). A), which influences the activity of a. Within the particular range of permeabilization conditions, objectives (1), (2), and (3) can all be understood through a set of permeabilization conditions; such conditions are high throughput. (HTS) assay is particularly useful. Here, various combinations of two or more molecules known or suspected to affect the activity of particular organelles and organelle components, optionally affecting such molecules (eg, such It is desirable to study the effects of further combinations of one or more molecules known or suspected to enhance, decrease, or otherwise modulate the activity of such molecules.

Results shown above derived from cells exposed to calcium ions (Ca ²⁻ ) and RU-360 (inhibitor of mitochondrial calcium monotransporter) (FIG. 1
9) illustrates one embodiment of the assay of the present invention. Here, permeabilization, while the plasma membrane has lost its ability to maintain selective permeability, intracellular (
For example, a treatment in which the organelle) membrane maintains selective permeability. That is, Ca ²⁺ diffuses into the cytosol across permeabilized plasma membranes under appropriate permeabilization conditions, but mitochondrial Ca ²⁺ uptake is inhibited by RU-360 in a concentration-dependent manner. To be done. Therefore, the mitochondrial membrane was not permissive for CA ²⁺ diffusion and was clearly mediated by the activity of the calcium unitransporter.

Example 13 FRET-Based Assay for ΔΨ in Permeabilized Cells Using Reduced ET Molecule Concentration In this example, a FRET-based assay for ΔΨ using NAO and TMR was used for the ET donor molecule. And using a permeabilized cell essentially as described in Example 12, except that a reduced loading concentration of ET receptor molecule was used, and affecting the mitochondrial activation status. Demonstrated the effects of four known drugs. According to a non-limiting theory, the use of lower concentrations of NAO and TMR, which are ET donor and ET acceptor molecules, avoids potential self-quenching by potentiometric dyes, and cationic dyes allow mitochondrial matrix It also avoids the undesired consumption of ΔΨ when invading.

The FRET method using digitonin-permeabilized SH-SY5Y cells was essentially as described in Example 12 and FIG. 16 above, except where noted herein. is there. All drugs are Sigma (St. L.) unless otherwise stated.
ouis, MO). Briefly, 96-well assay plates containing SY5Y cells were loaded with 85 nM NAO, washed and FLIP.
Placed on the R ^™ machine. Monitor the readings of the initial instrument and, as above,
The sample integrity was confirmed. The cells were then co-permeabilized with 0.01% digitonin and labeled with the ET molecule TMR (156 nM) and equilibrated. Then, various concentrations of mitochondrial active compounds (oligomycin,
Boncrecic acid, nigericin, or ADP) was added, and instrument readings were collected as described above at 5-second intervals. After about 10 to 12 minutes, CCCP (0
． 5 μM) or Ca ²⁺ (35 μM or 50 μM),
The mitochondrial membrane potential was plunged and the indicated values obtained for an additional 5-10 minutes.

FIG. 21 shows the superimposed FRET RFU time course plots obtained when the mitochondrial active compound was spiked with various concentrations of oligomycin, a specific inhibitor of ATP synthase. . By way of non-limiting theory, the mitochondrial inner membrane hyperpolarization observed following exposure of permeabilized cells to oligomycin (FIG. 21A) shows normal ΔΨ consumption from inhibition of ATP synthase and via ADP phosphorylation. Resulting from the inhibition. Therefore, TMR-increased quenching of NAO was observed in the presence of oligomycin in a dose-dependent manner between 5 and 12 minutes (EC50 = 0.25 μg / ml). Figure 21B shows the concentration of each oligomycin (data points ± SEM
, R ² = 0.4123) is calculated by calculating R−S as described in Example 12 above.

[0296] Following a similar reasoning, excess AD of permeabilized cells in the FRET assay.
Exposure to P should result in a transient decrease in ΔΨ when the mitochondrial potential membrane is consumed by ATP synthase-mediated phosphorylation of ADP. Such transient loss of ΔΨ was observed by the added mitochondrial active compound at 6.25 to 50 μM.
It was observed when it was ADP (FIG. 22A, EC50 = 12.1 μM). 6.
At less than 25 μM, the change in ΔΨ is buffer alone (ANOVA F =
It was indistinguishable from the control group exposed to 36.4). The CRC plot as a function of ADP concentration is shown in Figure 22B.

FIG. 23 shows the superimposes obtained when the mitochondrial active compound was supplemented with various concentrations of boncrekinic acid (BAK), a specific inhibitor of mitochondrial adenine nucleotide transferase.
d) shows a FRET RFU time course plot. As a non-limiting theory, the mitochondrial inner membrane hyperpolarization observed following exposure of permeabilized cells to BKA (Fig. 2).
3A) from inhibition of ADP entry into the mitochondrial matrix and from AD
It results from inhibition as a result of normal ΔΨ consumption via P phosphorylation. BKA is also believed to proactively prevent mitochondrial permeability changes resulting from Ca ²⁺ loading. Therefore, TMR-enhanced NAO quenching was observed in the presence of BKA in a dose-dependent manner between 5 and 11 minutes (EC50 = 0.09 μg / ml). After addition of Ca ²⁺ to permeabilized cells, BKA enhanced the extent of ΔΨ recovery and also mitigated the secondary loss of ΔΨ, probably due to permeability changes (FIG. 23).
A). FIG. 23B shows each BKA concentration (data points ± SEM, R ² = 0.58).
(Denoted by 22) shows the CRC produced by calculating R-S as described in Example 12 above.

FIG. 24 shows various concentrations of nigericin (proton-motive forc of about 220 mV across the mitochondrial membrane derived from a pH gradient).
e) Superimposed, obtained when the mitochondrial active compound was added with a specific potassium / proton exchanger that causes a part of the e) to plunge.
FIG. 6 shows an imposed) FRET RFU time course plot. By way of non-limiting theory, the mitochondrial inner membrane hyperpolarization observed following exposure of permeabilized cells to nigericin (FIG. 24A) is due to the electrochemical component of mitochondrial membrane proton transport (eg, electron transport). The resulting intact mechanism results from compensation for the loss of pH gradient. Therefore, the extinction of NAO increased by TMR is
It was observed in the presence of 0.09-0.75 μM nigericin at time points between 6 and 12 minutes. At higher nigericin concentrations (eg> 3 μM), there was a steep loss of NAO quenching (FIG. 24A), probably (and according to the unconstrained theory) that electron transport could not compensate for ΔpH consumption. caused by. FIG. 24B, for each nigericin concentration, determined RFU and described the FRET assay conditions (data points, ± SE, ANOVA P <0.0005 for data points, as described in Example 12 above. Shows the bar graphs produced by determining the concentration at which ΔΨ fell sharply (> 1.5 μM) and the concentration at which mitochondrial inner membrane hyperpolarization was detectable (<0.75 μM).

From the above, while particular embodiments of the present invention have been described herein for purposes of illustration, various modifications can be made without departing from the spirit and scope of the invention. That is clear. Accordingly, the invention is not limited except by the appended claims.

[Brief description of drawings]

FIG. 1 schematically illustrates direct and indirect methods for measuring energy transfer. Symbol: “λ _EX ”, peak excitation wavelength; “λ _EM ”, peak emission wavelength; “e
, Energy; white squares, receptive filter set; black squares, closed filter set.

Figure 2: Fractions designated as follows: "CS", cytosolic space; "OM",
Outer membrane; "IS", intermembrane space; "IM", intimal; "MX", the matrix; "D _MX"
A matrix compound, a donor compound localized in the matrix; "A _IM ", an acceptor compound localized in the inner membrane; " _DIS, " a donor compound localized in the intermembrane space; "e", energy, 2 schematically shows the sub-mitochondrial structural fraction and the energy transfer interaction between the energy transfer donor molecule and the energy transfer acceptor molecule.

FIG. 3 shows representative data from a FRET-based assay for ΔΨ _m . FIG. 3A shows data from a Type I assay; FIG. 3B shows data from a Type II assay.

FIG. 4 shows titration of ET donor molecule (NAO) and ET acceptor molecule (TMRM) in a ΔRET _m FRET assay.

FIG. 5 shows calibration of ET donor molecule (NAO) and ET acceptor molecule (TMRM) concentrations in a ΔΨ _m FRET assay.

FIG. 6 shows time course data from a ΔRET _m FRET assay using NAO and TMRM alone, and combinations thereof.

FIG. 7 shows a Type I ΔFRETΨ _m assay using various agents. symbol:"
MO ", medium (HBSS) only;" C ", CCCP;" I ", ionomycin;
"I + BKA", ionomycin, and bongrechicic acid
acid).

FIG. 8 shows a Type I FRETΔΨ _m assay for various agents. Symbol: "MO",
Medium (HBSS) only; "C", CCCP; "I + RR", ionomycin and ruthenium red.

FIG. 9 shows a Type I FRETΔΨ _m assay for various agents. Symbols: "MO", medium (HBSS) only; "I", ionomycin; "I + CsA", ionomycin and cyclosporin A. Vertical lines indicate standard error for each reading.

FIG. 10 is a dose response curve for the ΔΨ _m plummet drug CCCP.

FIG. 11 shows a Type II FRETΔΨ _m assay. Symbol: "MO", medium (H
Results from samples treated with BSS) only; "4BA;" Results from samples treated with the [Delta] [Psi] _m consumer 4-bromo-A23187; "C" and arrow indicate the time of CCCP addition to the samples. Indicates.

FIG. 12 is a dose response curve for a ΔΨ _m consuming compound (ionomycin).

FIG. 13 is a dose response curve for a compound (cyclosporin A) that protects mitochondria against the ΔΨ _m consuming compound (ionomycin).

FIG. 14 shows dose response curves of three cell lines to the ΔΨ _m consumer A-23187.

FIG. 15 shows FRET in cancer cells after experimentally induced loss of mitochondrial membrane potential.

FIG. 16 shows the concentration-dependent response of permeabilized cells to calcium ions leading to a ΔΨ collapse at higher concentrations of Ca ²⁺ .

FIG. 17 shows the same data shown in FIG. 16, where the mean values are plotted without error bars. Also, the response of permeabilized cells to CCCP (a drug known to induce a ΔΨ plunge) is not shown in FIG. 16, but FIG.
Shown in. It should be noted that at a concentration of 100 μM, Ca ²⁺ induces a plunge in ΔΨ, both in the extent of response and in the time course, in much the same manner as seen in cells treated with CCCP. is there.

FIG. 18 is a Ca ²⁺ concentration response curve (CRC) in permeabilized cells generated from the data shown in FIGS. 16 and 17.

FIG. 19 is a CRC of RU-360 in permeabilized cells also in contact with Ca ²⁺ . RU-360 is an inhibitor of mitochondrial calcium uniporter.

FIG. 20: Cyclosporin A CRC in permeabilized cells also in contact with Ca ²⁺ . Cyclosporin A is a drug known to regulate Ca ²⁺ -induced ΔΨ plunges.

FIG. 21 shows the CRC of oligomycin in permeabilized SH-SY5Y cells. Oligomycin is a specific ATP synthase inhibitor.

FIG. 22 shows CRC of ADP in permeabilized SH-SY5Y cells.

FIG. 23. C of voncrecic acid in permeabilized SH-SY5Y cells.
Indicates RC.

FIG. 24 shows nigericin (nig) in permeabilized SH-SY5Y cells.
ericin) CRC.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/542 G01N 33/542 33/58 33/58 Z // G01N 21/64 21/64 Z (31 ) Priority claim number 60 / 176,383 (32) Priority date January 14, 2000 (January 14, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, M , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gauche, Samitra S. United States California 92129, San Diego, Patos lane 12334 F-term (reference) 2G043 AA03 BA16 CA04 EA01 FA06 GA07 GB21 KA03 KA05 LA01 2G045 CB01 FA29 GC15 GC18 4B063 QA01 QA18 QA19 QA20 QQ02 QQ04 QQ08 QQ09 QQ20 QR48 QR66 QR69 QR77 QR78 QS12 QX01 QX05

Claims (107)

[Claims] 1. A method for assaying mitochondrial membrane potential comprising:
The method comprises the following steps: (a) a sample containing one or more mitochondria, either simultaneously or sequentially in either order, with a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the mitochondria. Contacting each of the energy transfer molecules, wherein: (i) the first energy transfer molecule and the second energy transfer molecule are at the same submitochondrial site or are receptively adjacent submitochondria. A site, each independently of each other, wherein the site is selected from the group consisting of outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) said first energy transfer molecule is An energy donor molecule, and the second energy transfer molecule is an energy acceptor molecule (B) exciting the energy donor molecule to produce an excited energy donor molecule; and (c) transferring energy from the first energy transfer molecule to the second energy transfer molecule. Detecting the signal generated, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential. 2. The method of claim 1, wherein the excited energy donor molecule transfers energy to the energy acceptor molecule to produce an excited energy acceptor molecule, And the signal detected in step (c) results from the energy released by the excited energy acceptor molecule. 3. The method of claim 1, wherein energy transfer from the first energy transfer molecule to the second energy transfer molecule results in a decrease in the detection signal. 4. The method of claim 1, further comprising contacting the mitochondria with an agent that induces consumption of mitochondrial membrane potential. 5. The method of claim 4, wherein the agent that induces consumption of mitochondrial membrane potential is an ionophore. 6. The method according to claim 1, further comprising the step of contacting the mitochondria with an agent that induces a sudden drop in mitochondrial membrane potential. 7. The method according to claim 6, wherein the agent that induces a sharp drop in mitochondrial membrane potential is selected from the group consisting of CCCP and FCCP. 8. The method of claim 1, wherein the sample is washed prior to the step of detecting a signal. 9. The method of claim 1, wherein the signal detected in step (c) is compared to a reference signal. 10. The method according to claim 9, wherein the reference signal is an indicator of cell number, an indicator of mitochondrial mass, an indicator of cellular protein, an indicator of cellular DNA, an indicator of mitochondrial DNA, an indicator of mitochondrial protein. And a fluid volume indicator produced by an indicator selected from the group consisting of: 11. The method of claim 1, wherein the sample comprises one or more mitochondria present in at least one cell and the signal detected in step (c) is a reference. The method that is compared to the signal. 12. The method of claim 11, wherein the reference signal is
A method produced from a subcellular site selected from the group consisting of outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space, mitochondrial matrix, cytoplasm, nucleus, nuclear membrane and plasma membrane. 13. The method of claim 11, wherein the reference signal is produced from extracellular medium. 14. The method of claim 1, wherein the mitochondria are present in at least one cell during at least one step. 15. The method of claim 14, wherein the cell is an organism. 16. The method of claim 14, wherein the cells are cultured cells. 17. The method of claim 14, wherein the cells are cytoplasmic hybrid cells. 18. The method of claim 14, wherein the cells are plant cells. 19. The method of claim 14, wherein the cells are animal cells. 20. The method of claim 14, wherein the cells are present in a biological sample from a multicellular organism. 21. The method of claim 20, wherein the cells are plant cells. 22. The method of claim 20, wherein the cells are animal cells. 23. The method of claim 22, wherein the animal is a mammal. 24. The method of claim 23, wherein the mammal is a human. 25. The human being has, is suspected of, or is at risk of having, a disease or disorder associated with organelle dysfunction.
The method according to 4. 26. The method of claim 25, wherein the organelle dysfunction is mitochondrial dysfunction. 27. The method of claim 25, wherein the organelle dysfunction is lysosomal dysfunction. 28. The method of claim 1, wherein the first energy transfer molecule is located at a sub-mitochondrial site selected from the group consisting of a mitochondrial matrix and an inner mitochondrial membrane, and the second The method, wherein the energy transfer molecule is located at a sub-mitochondrial site selected from the group consisting of mitochondrial matrix and inner mitochondrial membrane. 29. The method of claim 28, wherein the concentration of the first energy transfer molecule at the sub-mitochondrial site does not change as a function of membrane potential and the second energy transfer of the mitochondrial matrix. The method wherein the concentration of is reduced as a function of membrane potential. 30. The method of claim 29, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 373 nm to about 390 nm and maximum emission at a wavelength of about 400 nm to about 500 nm. And (b) the second energy transfer molecule has maximum excitation at a wavelength of about 400 nm to about 500 nm. 31. The method according to claim 30, wherein (i) the first energy transfer molecule is a fusion protein, and the fusion protein is (a) Phe-64, Ser-65, Tyr. A blue-shifting green fluorescent protein polypeptide having a mutation in at least one of -66, Val-68 and Tyr-145, and (b) wherein the fusion protein is selected from the group consisting of mitochondrial matrix and inner mitochondrial membrane. (Ii) the second energy transfer molecule is DASPEI, DASPMI, 4;
-Di-1-ASP, 2-Di-1-ASP, DiOC ₇ (3), DiOC ₆ (3
), JC-1 and SYTO® 18 yeast mitochondrial strains. 32. The method of claim 29, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 425 nm to about 440 nm and maximum emission at a wavelength of about 450 nm to about 535 nm. And (b) the second energy transfer molecule has maximum excitation at a wavelength of about 450 nm to about 530 nm. 33. The method according to claim 32, wherein (i) the first energy transfer molecule is a fusion protein, and the fusion protein is (a) Phe-64, Ser-65, Tyr. -66, Asn-146
, Met-153, Val-163 and Asn-212
A blue-green-shifted green fluorescent protein polypeptide having a mutation in one and (b) a polypeptide sequence that positions the fusion protein at a sub-mitochondrial site selected from the group consisting of a mitochondrial matrix and an inner mitochondrial membrane; And (ii) the second energy transfer molecule is DASPEI, 2-Di-1-A
SP, DiOC ₆ (3), SYTO® 18 yeast mitochondrial strain,
A method selected from the group consisting of Rhodamine 6G, JC-1, NBD C6-ceramide and NBD C6-sphingomyelin. 34. The method of claim 29, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 470 nm to about 500 nm and maximum emission at a wavelength of about 505 nm to about 565 nm. And (b) the second energy transfer molecule has maximum excitation at a wavelength of about 505 nm to about 565 nm. 35. The method of claim 34, wherein (i) the first energy transfer molecule is nonyl acridine orange, Mit.
oTracker (registered trademark) Green FM, Mito Fluor ^™ Gr
een, and a fusion protein, wherein the fusion protein is (a) a wild-type green fluorescent protein polypeptide, Phe-64, Ser.
-65, Tyr-66, Gln-69, Ser-72 and Red-shifting green fluorescent protein polypeptide having a mutation in one or more of Thr-203, and Phe-64, Ser-65, Tyr-66. , A green fluorescent protein polypeptide selected from the group consisting of yellow-shifted green fluorescent protein polypeptides having a mutation in one or more of Gln-69, Ser-72 and Thr-203, and (b) the fusion. A polypeptide sequence that positions a protein at a sub-mitochondrial site selected from the group consisting of mitochondrial matrix and inner mitochondrial membrane; and (ii) said second energy transfer molecule is rhodamine 123, JC-1, tetrabromorhodamine 123, Rhodamine 6G, TMRM, TMRE, Tetra A method selected from the group consisting of methylosamin and rhodamine B. 36. The method of claim 29, wherein (a) the first energy transfer molecule has a maximum excitation at a wavelength of about 545 nm to about 560 nm and a maximum emission at a wavelength of about 565 nm to about 625 nm. And (b) the second energy transfer molecule has maximum excitation at a wavelength of about 565 nm to about 625 nm. 37. The method of claim 36, wherein (i) the first energy transfer molecule is MitoTracker® Orange CMTMRos; and (ii) the second energy transfer molecule is , DiOC ₂ (5). 38. The method of claim 29, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 495 nm to about 510 nm and maximum emission at a wavelength of about 510 nm to about 570 nm. And (b) the second energy transfer molecule has maximum excitation at a wavelength of about 510 nm to about 560 nm. 39. The method of claim 38, wherein (i) the first energy transfer molecule is a fusion protein and the fusion protein is (a) Ser-65, Tyr-66, Ser. -72 and Thr-
A polypeptide sequence selected from the group consisting of a FLASH protein sequence and a yellow-shifted green fluorescent protein polypeptide sequence having one or more mutations in 203; and (b) the fusion protein from the mitochondrial matrix and inner mitochondrial membrane. (Ii) the second energy transfer molecule comprises JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylosamine, a polypeptide sequence located at a submitochondrial site selected from the group consisting of: , Rhodamine B and 4-dimethylamino-tetramethylosamine. 40. The method of claim 1, wherein the relative amount of the signal produced by energy transfer is detected. 41. The method of claim 1, wherein the signal is detected over a period of time and the rate of change of the signal level is measured. 42. The method of claim 1, wherein the signal is detected and integrated over a period of time. 43. The method of claim 1, wherein the membrane potential comprises a potential, a pH potential or both. 44. The method of claim 1, wherein the first energy transfer molecule and the second energy transfer molecule are located within a range of about 10Å to about 100Å with respect to each other. 45. The method of claim 1, wherein the first energy transfer molecule and the second energy transfer molecule are located within a range of about 10Å to about 50Å with respect to each other. 46. The method of claim 1, wherein the first energy transfer molecule and the second energy transfer molecule are located within the range of about 20Å to about 50Å with respect to each other. 47. The method of any one of claims 1-46, wherein the signal is generated by fluorescence resonance energy transfer. 48. A method for identifying an agent that modifies a mitochondrial membrane potential, the method comprising the steps of: (a) selecting one or more mitochondria in the absence and presence of a candidate agent. Contacting the sample containing with each of the first energy transfer molecule and the second energy transfer molecule that are not endogenous to the mitochondria, either simultaneously or sequentially, where: (I) The first energy transfer molecule and the second energy transfer molecule are each independently located at the same submitochondrial site or at an acceptably adjacent submitochondrial site, wherein the site is a mitochondrial site. Selected from the group consisting of outer membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix, and (i i) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (b) exciting and exciting the energy donor molecule Generating an energy donor molecule; (c) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, the energy transfer in the mitochondria The concentration of at least one of the molecules changes as a function of membrane potential; and (d) the signal produced in the absence of the candidate agent and the signal produced in the presence of the candidate agent. And then identifying an agent that modifies the cell membrane potential. 49. A method for identifying a regulator of a drug that alters mitochondrial membrane potential, which method comprises the steps of: (a) in the absence and presence of a candidate regulator, (1) mitochondria. 49. An agent selected from the group consisting of agents that alter membrane potential and agents identified according to the method of claim 48, and (2) a sample containing one or more mitochondria, either simultaneously or sequentially in either order. And contacting each of the first energy transfer molecule and the second energy transfer molecule that are not endogenous to the mitochondria, wherein: (i) the first energy transfer molecule and the second energy transfer molecule; The two energy transfer molecules are independent of each other at the same submitochondrial site or at an acceptably adjacent submitochondrial site. Each of which is located and the site is selected from the group consisting of outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) the first energy transfer molecule is an energy donor molecule. And and the second energy transfer molecule is an energy acceptor molecule; (b) exciting the energy donor molecule to produce an excited energy donor molecule; (c) the first Detecting the signal generated by the energy transfer from the energy transfer molecule to the second energy transfer molecule, the concentration of at least one of the energy transfer molecules in the mitochondria as a function of membrane potential. Changing steps; and (d) the sequence produced in the absence of the candidate regulator. Comparing the signal to the signal produced in the presence of the candidate regulator, and then identifying the regulator of the agent that modifies the mitochondrial membrane potential. 50. The method of claim 49, wherein the regulator is an agonist of a drug that modifies mitochondrial potential. 51. The method of claim 49, wherein the regulator is an antagonist of a drug that modifies mitochondrial potential. 52. The method of claim 49, wherein the agent that modifies the mitochondrial potential is an apoptogen. 53. The method of claim 49, wherein the agent that modifies the mitochondrial potential is selected from the group consisting of thapsigargin, ionophores and excitatory amino acids or derivatives thereof. 54. The method of claim 53, wherein the ionophore is selected from the group consisting of ionomycin and A23187. 55. The method of claim 53, wherein the excitatory amino acid or derivative thereof is selected from the group consisting of glutamic acid, NAAG, NMDA, AMPA, APPA and kainic acid. 56. An agent that preferentially modifies mitochondrial membrane potential in mitochondria from a first biological source and does not substantially modify mitochondrial membrane potential in mitochondria from a second biological source. A method for identifying, comprising the steps of: (a) a first biological sample and a second organism comprising one or more mitochondria in the absence and presence of a candidate agent. Contacting each of the biological samples with each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the mitochondria, either simultaneously or sequentially, Where: (i) the first sample is from a first biological source, and the second sample is a second organism different from the first biological source. (Ii) the first energy transfer molecule and the second energy transfer molecule are each independently located at the same submitochondrial site or at an acceptably adjacent submitochondrial site, independent of one another. , The site is selected from the group consisting of outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix, and (iii) said first energy transfer molecule is an energy donor molecule and said second The energy transfer molecule of is an energy acceptor molecule; (b) Exciting the energy donor molecule in the presence of each of the first sample and the second sample to produce an excited energy donor Producing a molecule; (c) the first sample in the presence of each of the first sample and the second sample. Detecting the signal generated by the energy transfer from the energy transfer molecule to the second energy transfer molecule, the concentration of at least one of the energy transfer molecules in the mitochondria as a function of membrane potential. Changing; and (d) the signal produced in the presence of each of the first sample and the second sample and in the absence of the candidate agent, the first sample and the Comparing the signal generated in the presence of each of the second samples and in the presence of the candidate agent, and then identifying an agent that preferentially modifies mitochondrial membrane potential. . 57. The method of claim 56, wherein the first biological source and the second biological source are different species. 58. The method of claim 56, wherein the first biological source is suspected of having a disease, diagnosed as having a disease, or susceptible to having a disease. A method wherein the mammal is a mammal and the second biological source is not suspected of having the disease and has not been diagnosed as having the disease and is not susceptible to the disease. . 59. The method of claim 58, wherein the first biological source is human and the second biological source is human. 60. The disease is Alzheimer's disease, Parkinson's disease and II.
59. The method of claim 58, selected from the group consisting of type 2 diabetes. 61. An agent is identified that preferentially modifies mitochondrial membrane potential in mitochondria from a first biological sample and does not substantially modify mitochondrial membrane potential in mitochondria from a second biological sample. A method for producing a first biological sample and a second biological sample comprising one or more mitochondria in the absence and presence of a candidate agent. Contacting each of the samples with each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the mitochondria, either simultaneously or sequentially in either order, wherein (I) the first sample is from a first tissue and the second sample is from a second biological tissue that is different from the first tissue. (Ii) the first energy transfer molecule and the second energy transfer molecule are each independently located at the same submitochondrial site or at an acceptably adjacent submitochondrial site, the site being outside the mitochondria. A membrane, an inner mitochondrial membrane, a mitochondrial intermembrane space and a mitochondrial matrix, and (iii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule. A body molecule; (b) exciting the energy donor molecule in the presence of each of the first sample and the second sample to produce an excited energy donor molecule; c) the first energy transfer component in the presence of each of the first sample and the second sample. Detecting a signal generated by energy transfer from a child to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential, And (d) the signal generated in the presence of each of the first sample and the second sample and in the absence of the candidate agent, the first sample and the second sample. Comparing the signal generated in the presence of each of the samples and in the presence of the candidate agent, and then identifying an agent that preferentially modifies mitochondrial membrane potential. 62. The method of claim 61, wherein the first tissue and the second tissue are obtained from the same subject. 63. The method of claim 61, wherein the first tissue and the second tissue are each obtained from a subject of the same species. 64. The method of claim 61, wherein the first tissue and the second tissue are obtained from subjects of different species. 65. A method of detecting a fusion of a first mitochondria and a second mitochondria, the method comprising the steps of: (a) a first sample containing one or more mitochondria; Contacting with a first energy transfer molecule that is not endogenous to the mitochondria; (b) a second sample containing one or more mitochondria with a second energy transfer molecule that is not endogenous to the mitochondria. Contacting; wherein (i) the first energy transfer molecule and the second energy transfer molecule are each independently located at the same submitochondrial site or at an acceptably adjacent submitochondrial site, independent of each other. The site consists of the outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix. (Ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (c) the first sample is selected from the group: Contacting with the second sample under conditions and for a time sufficient to allow mitochondrial fusion; (d) exciting the energy donor molecule to produce an excited energy donor molecule; (E) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule and then determining the fusion of the first and second mitochondria. , Including. 66. A method of identifying an agent that alters mitochondrial fusion, the method comprising the steps of: (a) a first sample containing one or more mitochondria endogenous to said mitochondria. Contacting with a first energy transfer molecule that is not endogenous; (b) contacting a second sample containing one or more mitochondria with a second energy transfer molecule that is not endogenous to the mitochondria; , (I) the first energy transfer molecule and the second energy transfer molecule are each independently located at the same submitochondrial site or at an acceptably adjacent submitochondrial site, each of which is Selected from the group consisting of outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space and mitochondrial matrix, And (ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (c) in the absence and presence of a candidate agent. Contacting the first sample with the second sample under conditions and for a time sufficient to allow mitochondrial fusion; (d) exciting the energy donor molecule to produce an excited energy donor. Generating a molecule; (e) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule; and (f) in the absence of the candidate agent. Comparing the detected signal with the detected signal in the presence of the candidate agent, and then identifying an agent that modifies the mitochondrial fusion. The, way. 67. The agent of claim 4, wherein the agent increases mitochondrial membrane potential.
The method according to any one of 8, 49, 56, 61 or 66. 68. The agent of claim 48, wherein the agent consumes mitochondrial membrane potential.
The method of any one of claims 49, 56, 61 or 66. 69. The agent of claim 4, wherein the agent causes a rapid drop in mitochondrial membrane potential.
The method according to any one of 8, 49, 56, 61 or 66. 70. The method of any one of claims 48, 49, 56, 61 or 66, wherein the agent modifies the equilibrium distribution of at least one ionic species on either side of the cell membrane. 71. The method of claim 70, wherein the ionic species is Ca ²⁺ and the cell membrane is a mitochondrial membrane. 72. The method of claim 69, wherein the agent that causes a sudden drop in mitochondrial membrane potential is an apoptogen. 73. The method of claim 69, wherein the agent that plunges the mitochondrial membrane potential interacts with an adenine nucleotide translocator. 74. The method of claim 73, wherein the agent that causes a drop in mitochondrial membrane potential is selected from the group consisting of atractyloside, carboxyatractyloside, bongcrecic acid, and isobocclecic acid. 75. A reagent for measuring mitochondrial Δψ comprising a FRET donor molecule and a FRET acceptor molecule, wherein the accumulation of at least one of said molecules in mitochondria is Δψ. , And the other accumulation of the molecule in mitochondria is independent of Δψ. 76. The reagent according to claim 75, wherein the molecule that accumulates in mitochondria independent of Δψ is NAO, MitoTracker (registered trademark) Green FM, MitoFluor ^™ , DAPI, and the following (a):
And a fusion protein comprising (b): (a) a polypeptide selected from the group consisting of a red-shifted green fluorescent protein polypeptide, a yellow-shifted green fluorescent protein protein and a "FLASH" polypeptide, and (B) a polypeptide sequence that positions the fusion protein in the mitochondrial matrix or inner mitochondrial membrane, a reagent selected from the group consisting of: 77. The reagent according to claim 75 or 76,
Molecules that accumulate in mitochondria in a manner dependent on Δψ are TMRM, TM
RE, rhodamine 123, ethidium bromide, 4-Di-1-ASP, 2-Di-
1-a reagent selected from the group consisting of ASP and DASPEI. 78. A kit comprising the reagent of claim 75 and an auxiliary reagent for measuring mitochondrial Δψ. 79. A method for assaying cell membrane potential, the method comprising the steps of: (a) a sample comprising at least one cell membrane, either simultaneously or sequentially in either order. Contacting each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to, wherein: (i) the first energy transfer molecule and the second energy transfer. The molecules are each located independently of one another at the same membrane site, or at an acceptably adjacent membrane site, such that at least one of the energy transfer molecules is located at a cell membrane forming a subcellular compartment, And (ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; Exciting the energy donor molecule to produce an excited energy donor molecule; and (c) a signal produced by energy transfer from the first energy transfer molecule to the second energy transfer molecule. Detecting, wherein the concentration of at least one of the energy transfer molecules at the membrane site changes as a function of membrane potential. 80. The method of claim 79, wherein the first energy transfer molecule is located at a first membrane site selected from the group consisting of mitochondria, endoplasmic reticulum, Golgi, lysosomes and plasma membranes. And the second energy transfer molecule is located at the same membrane site or at an acceptably adjacent membrane site selected from the group consisting of mitochondria, endoplasmic reticulum, Golgi, lysosomes and plasma membranes. 81. The method of claim 79, wherein the concentration of said first energy transfer molecule at said first membrane site does not change as a function of membrane potential and said first site at said membrane site. The method, wherein the concentration of the energy transfer molecule of 2 decreases as a function of membrane potential. 82. The method of claim 79, wherein: (a) the first energy transfer molecule has maximum excitation at a wavelength of about 373 nm to about 390 nm and a wavelength of about 400 nm to about 500 nm. And (b) the second energy transfer molecule has a maximum excitation at a wavelength of about 400 nm to about 500 nm. 83. The method of claim 79, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 425 nm to about 440 nm and at a wavelength of about 450 nm to about 535 nm. Has a maximum emission; and (b) the second energy transfer molecule has a maximum excitation at a wavelength of about 450 nm to about 530 nm. 84. The method of claim 79, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 470 nm to about 500 nm and at a wavelength of about 505 nm to about 565 nm. Having a maximum emission; and (b) the second energy transfer molecule has a maximum excitation at a wavelength of about 505 nm to about 565 nm. 85. The method of claim 79, wherein (a) the first energy transfer molecule has maximum excitation at a wavelength of about 545 nm to about 560 nm and at a wavelength of about 565 nm to about 625 nm. Having a maximum emission; and (b) the second energy transfer molecule has a maximum excitation at a wavelength of about 565 nm to about 625 nm. 86. A method for identifying an agent that alters a cell membrane potential, the method comprising the steps of: (a) including one or more cell membranes in the absence and presence of a candidate agent. Contacting the sample with each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the sample, either simultaneously or sequentially, where: ( i) The first energy transfer molecule and the second energy transfer molecule are each located independently of one another at the same membrane site or at an acceptably adjacent membrane site, so that At least one of which is located in a cell membrane forming a subcellular compartment, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is A step of (b) exciting the energy donor molecule to produce an excited energy donor molecule; (c) the first energy transfer molecule to the second energy transfer. Detecting a signal generated by energy transfer to a molecule, wherein the concentration of at least one of said energy transfer molecules in said subcellular compartment changes as a function of membrane potential; and (d). Comparing the signal produced in the absence of the candidate agent with the signal produced in the presence of the candidate agent, and then identifying an agent that alters cell membrane potential. 87. A method for identifying a regulator of a drug that alters cell membrane potential, which method comprises the steps of: (a) in the absence and presence of a candidate regulator, (1) cell membrane potential. 89. An agent selected from the group consisting of: an agent that modifies a. And an agent identified according to the method of claim 86; and (2) a sample containing one or more cell membranes, either simultaneously or sequentially in any order, Contacting each of a first energy transfer molecule and a second energy transfer molecule that is not endogenous to the sample, wherein: (i) the first energy transfer molecule and the second energy transfer molecule; The energy transfer molecules are each located at the same membrane site or at an acceptably adjacent membrane site independently of each other, such that at least one of the energy transfer molecules is One is located at the cell membrane forming a subcellular compartment, and (ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; b) exciting the energy donor molecule to produce an excited energy donor molecule; (c) a signal produced by energy transfer from the first energy transfer molecule to the second energy transfer molecule. Wherein the concentration of at least one of the energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and (d) is produced in the absence of the candidate regulator. Of the agent that modifies the cell membrane potential from the signal compared to the signal generated in the presence of the candidate regulator. Identifying the regulator. 88. An agent is identified that preferentially modifies the cell membrane potential in a membrane from a first biological source and does not substantially modify the cell membrane potential in a membrane from a second biological source. A method for producing a first biological sample and a second biological sample comprising one or more cell membranes in the absence and presence of a candidate agent. Contacting each of the samples, either simultaneously or sequentially in either order, with each of the first energy transfer molecule and the second energy transfer molecule that is not endogenous to the sample, wherein (I) the first sample is from a first biological source and the second sample is a second biological source that is different from the first biological source. And (ii) the first energy transfer molecule and And the second energy transfer molecule are each independently located at the same membrane site or at an acceptably adjacent membrane site independent of each other such that at least one of the energy transfer molecules forms a subcellular compartment. (Iii) the first energy transfer molecule is an energy donor molecule, and the second energy transfer molecule is an energy acceptor molecule; and (b) the first energy transfer molecule is an energy donor molecule. Exciting the energy donor molecule in the presence of each of a sample and the second sample to produce an excited energy donor molecule; (c) the first sample and the second sample. Detecting the signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule in the presence of each of the sub-cells. The concentration of at least one of the energy transfer molecules in the cell compartment changes as a function of membrane potential; and (d) in the presence of each of the first sample and the second sample, and Comparing the signal generated in the absence of a candidate agent with the signal generated in the presence of each of the first sample and the second sample and in the presence of the candidate agent, And then identifying an agent that preferentially modifies the cell membrane potential. 89. For identifying an agent that preferentially modifies the cell membrane potential in a membrane derived from a first biological sample and does not substantially alter the cell membrane potential in a membrane derived from a second biological sample. A method comprising: (a) a first biological sample and a second biological sample comprising one or more cell membranes in the absence and presence of a candidate agent. Contacting each with either each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the sample, either simultaneously or sequentially, where: ( i) the first sample is from a first tissue and the second sample is from a second tissue different from the first tissue; (ii) the first energy transfer molecule And a second energy transfer molecule Are each located independently of one another at the same membrane site or at an acceptably adjacent membrane site such that at least one of the energy transfer molecules is located at a cell membrane forming a subcellular compartment, and (Iii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (b) the first sample and the second sample Exciting each of the energy donor molecules in the presence of each of, to produce excited energy donor molecules; (c) in the presence of each of the first sample and the second sample, Detecting a signal generated by energy transfer from a first energy transfer molecule to the second energy transfer molecule, said energy in said subcellular compartment The concentration of at least one of the transfer molecules changes as a function of membrane potential; and (d) in the presence of each of the first sample and the second sample and in the absence of the candidate agent. Comparing the signal generated below with the signal generated in the presence of each of the first sample and the second sample and in the presence of the candidate agent, and then prioritizing cell membrane potential Identifying the agent that chemically modifies. 90. A method for detecting a particular type of cell in a sample, the method comprising the steps of: (a) a sample containing one or more mitochondria, either simultaneously or sequentially. In order of contacting each of a first energy transfer molecule and a second energy transfer molecule that are not endogenous to the mitochondria, wherein: (i) the first energy transfer molecule And the second energy transfer molecule is located independently of each other at the same subcellular site or at an acceptably adjacent subcellular site, and (ii) the first energy transfer molecule is an energy donor. A molecule, and the second energy transfer molecule is an energy acceptor molecule, step; (b) exciting the energy donor molecule to produce an excited energy donor moiety. And (c) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, wherein at least one of the energy transfer molecules is included. One preferentially accumulating in the particular type of cells, wherein the signal correlates with the presence of the particular type of cells in the sample. 91. The method of claim 90, wherein the method compares the signal generated in the sample with a signal generated from a control sample lacking the particular type of cells. The method further comprising: 92. The method of claim 90, wherein the particular type of cell is a cancer cell. 93. A method for identifying a Δψ _m stabilizer comprising the steps of: (a) (1) Δψ in the absence and presence of a candidate Δψ _m stabilizer. An agent that modifies _m and (2) a sample containing one or more mitochondria, together with a first energy transfer molecule and a second energy transfer that are not endogenous to the mitochondria, either simultaneously or sequentially. Contacting each of the molecules, wherein: (i) the first energy transfer molecule and the second energy transfer molecule are:
Each is located independently of one another at the same sub-mitochondrial site or at an acceptably adjacent sub-mitochondrial site, which site is selected from the group consisting of the outer mitochondrial membrane, the inner mitochondrial membrane, the mitochondrial intermembrane space and the mitochondrial matrix. And (ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (b) exciting the energy donor molecule Generating an excited energy donor molecule; (c) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, the mitochondria The concentration of at least one of the energy transfer molecules in Varying as a function; and (d) the signal generated in the absence of the candidate Δψ _m stabilizer and the candidate Δψ _m.
comparing the signal generated in the presence of the ψ _m stabilizer and then identifying the Δψ _m stabilizer. 94. The method of claim 93, wherein said mitochondria are contained intracellularly. 95. The method of claim 94, wherein the agent that modifies Δψ _m is an agent that increases levels of cytosolic Ca ²⁺ . 96. The agent of increasing the level of cytosolic Ca ²⁺ is selected from the group consisting of a calcium ionophore and thapsigargin.
The method according to 5. 97. The method of claim 95, wherein said cells contain one or more types of glutamate receptors. 98. The method of claim 97, wherein the agent that increases levels of cytosolic Ca ²⁺ is an excitatory amino acid or derivative thereof. 99. The method of claim 98, wherein the excitatory amino acid or derivative thereof is selected from the group consisting of glutamic acid, NAAG, NMDA, AMPA, APPA and kainic acid. 100. A Δψ _m stabilizer identified according to the method of claim 99. 101. A method of treating seizures, the Δ of claim 100.
A method comprising administering a ψ _m stabilizer to a patient in need thereof. 102. The method of claim 14, wherein the cells are permeabilized cells. 103. The method of claim 20, wherein the cells are permeabilized cells. 104. The method of claim 94, wherein said cells are permeabilized cells. 105. The reagent of claim 77, wherein the first FRET molecule that accumulates in the mitochondria is contacted with a cell population in aqueous solution at 0.0
The second molecule, which is formulated to lyse the population of cells in the aqueous solution to the extent necessary to saturate the first molecule within 1-2 minutes, and accumulates in the mitochondria A reagent that is formulated to lyse to a degree necessary to saturate the cell population in the aqueous solution with the second molecule within 2.5 to about 5 minutes of contacting the cell population. 106. The reagent of claim 77, wherein one of the mitochondrial accumulating molecules is dissolved in an aqueous solution and the other of the mitochondrial accumulating molecules is present in the reagent in solid form. 107. The reagent of claim 106, wherein the molecule that accumulates in the mitochondria and is present in solid form in the reagent is 0.01 min to about 0.01 min after contact with the cell population in aqueous solution. The cell population in the aqueous solution is added to the second within 5 minutes.
Reagents that are formulated to dissolve to the extent necessary to saturate the molecules of.