HK1162664B - Identifying molecules that modulate protein- protein interactions using protease activated reporters - Google Patents

Description

Identification of molecules that modulate protein-protein interactions using protease-activated receptors

Technical Field

The present invention relates to materials and methods for determining the interaction between molecules of interest. More particularly, the invention relates to determining whether a particular substance, such as a "test compound", modulates the interaction between two or more particular proteins of interest. Determining the interaction between proteins involves monitoring the activation of a reporter molecule, which may be present in the cell, in solution, or in an artificial package or unit containing one or more reactants of interest, wherein the genetic activation or inactivation of the reporter is dependent on whether or not a modulating effect occurs. Such determinations are typically achieved using transformed or transfected cells. These transformed or transfected cells, as well as reagents for transforming or transfecting cells, are also an aspect of the present invention. In addition, a cell-free system or a system utilizing artificial packages or units, such as viruses, virus-like particles, liposomes, etc., carrying one or more agents of interest may also be employed.

Background of the invention and related Art

The study of protein-protein interactions through the identification of receptor ligands is a field of great interest. Even if one or more ligands for a particular receptor are known, it would be desirable to identify more potent or selective ligands. The G-protein coupled receptor GPCR, also known as the seven transmembrane receptor (7TMR), will be discussed herein as a non-exclusive example of a class of proteins that can be characterized in this manner. However, any protein capable of interacting, such as a member of a metabolic pathway or a cascade, is suitable for use in the present assay.

GPCRs are the largest class of cell surface receptors known to humans and are therefore considered to be the main subject of application of the present invention. Ligands that modulate GPCR signaling include hormones, neurotransmitters, peptides, glycoproteins, lipids, nucleotides, and ions. GPCRs are also known as sensory receptors, e.g., receptors for light, smell, pheromone, and taste. Because GPCRs have such a large and diverse role, they are the subject of many active studies, for example, studies in chemical warfare defense and biological warfare defense applications, and in the field of drugs for the treatment of various disorders. Many drugs have been successfully discovered. For example, Howard et al estimate that more than 50% of commercially available drugs act on these receptors (Howard, et al, Trends pharmacol. sci., 22: 132140 (2001)).

As used herein, "GPCR" refers to any member of the GPCR receptor superfamily. This superfamily is characterized by a seven-transmembrane domain (7TM) structure. Examples of such receptors include, but are not limited to, class a or "rhodopsin-like" receptors; class B or "secretin-like" receptors; class C or "metabotropic glutamate-like" receptors; frizzled and Smoothened related receptors; the adhesion receptor family or EGF-7TM/LNB-7TM receptors; adiponectin receptors and related receptors; and chemical sensory receptors, including olfactory, taste, vomeronasal, and pheromone receptors. For example, the human GPCR superfamily includes, but is not limited to, receptor molecules described in the following references: vasilatis, et al, proc.natl.acad.sci.usa, 100: 49034908 (2003); takeda, et al, FEBS Letters, 520: 97101 (2002); fredricksson, et al, mol. 12561272 (2003); glusman, et al, Genome res, 11: 685702 (2001); and Zozulya, et al, Genome biol., 2: 0018.10018.12(2001).

Briefly, the general mechanism of action of GPCR function is as follows: 1) GPCRs bind to ligands, 2) cause conformational changes, thereby 3) provoke a cascade of cellular events leading to changes in cellular physiology. GPCRs transduce signals by modulating the activity of a range of intracellular proteins, such as heterotrimeric guanine nucleotide binding proteins (G proteins) and β arrestins (β arrestins). In the case of G proteins, the ligand-receptor complex stimulates an exchange of guanine nucleotides and dissociates the heterotrimer of G proteins into α and β γ subunits. In other cases, the β -arrestin may replace the G-protein, counteract G-protein signaling, coordinate G-protein signaling, and the like.

Both GTP-bound alpha subunit and beta gamma heterodimer have been observed to modulate a variety of cellular effector proteins, including adenylyl cyclase and phospholipase c (plc). In conventional cell-based GPCR assays, receptor activity is monitored by measuring the output of G protein-regulated effector channels, such as the accumulation of cAMP produced by adenylyl cyclase or the release of intracellular calcium stimulated by PLC activity.

For a variety of reasons, it is difficult to develop conventional G-protein based signal transduction assays for certain subjects. First, for example, different GPCRs are coupled to different G protein-regulated signal transduction pathways. Conventional G-protein based assays rely on knowledge of the G-protein specificity of the target receptor, or the assay requires the construction of cell systems that force the target receptor to couple to a selected G-protein effector pathway. Second, since the GPCR superfamily is so large, all cells express many endogenous GPCRs (as well as other receptors and signaling factors). Thus, in addition to the target GPCR, the measured effector pathway can also be modulated by endogenous molecules. This phenomenon may lead to false positive or false negative results, for example when attempting to identify selective modulators of the GPCR of interest.

Modulation of G protein activity is not the only result of ligand/GPCR binding. See, e.g., Luttrell, et al, j.cell sci, l 15: 455465(2002), and Ferguson, pharmacol.rev., 53: 124(2001). These documents comment on activities that may lead to attenuation or termination of GPCR signal. These termination processes are useful for preventing cell overstimulation and for forcing a temporary connection between an extracellular signal and the corresponding intracellular pathway.

In general, binding of an agonist to a GPCR will result in phosphorylation of serine and threonine residues at the C-terminus of the receptor molecule by GPCR kinases. Agonists of agonist-complexed C-terminally phosphorylated GPCRs then interact with arrestin family members such as α arrestin, β arrestin or β arrestin 2, which down-regulate or inhibit receptor signal transduction. This binding can inhibit the coupling of the receptor to the G protein, thereby internalizing the receptor, followed by degradation and/or recycling. For example, binding of an arrestin, such as β arrestin 2, to a phosphorylated GPCR may decrease the activity of the target GPCR in different ways. The simplest mechanism by which an arrestin inhibits its target activation is to bind to the intracellular domain of the GPCR, thereby blocking the binding site of the heterotrimeric G protein and preventing extracellular signals from activating the signaling pathway (desensitization). Another regulatory mechanism used by arrestins is to link the receptor to elements of the membrane internalization machinery (e.g., clathrin-mediated endocytosis), which will cause the receptor to internalize within the envelope vesicle and fuse with the endosome. Once in the endosome, the receptor can be degraded (e.g., lysosomes) or can be recycled to the plasma membrane where it is activated again.

Thus, it can be said that binding of a ligand to a GPCR "modulates" the interaction between the GPCR and the arrestin, since binding of the ligand to the GPCR results in binding of the arrestin to the GPCR, thereby modulating its activity. In this context, "modulate" or any other form thereof, when referring to interaction or binding, refers only to certain changes in the manner of interaction between two proteins of the invention, for example, comparing how the two proteins interact in the presence and absence of a test compound or ligand. Thus, modulation involves the binding of only two molecules. For example, the presence of a test compound may potentiate or enhance, attenuate, block, inhibit, redirect, reduce, or alter the interaction between two proteins in some detectable manner or form, or the test compound may promote the likelihood of an interaction, and the like.

In some cases, 7TMR signaling may occur independently of G proteins. Thus, when a ligand binds to 7TMR, it is the β arrestin that is recruited to participate or initiate the amplification of the signaling cascade in the cell, rather than the G protein. See, e.g., Violin & Lefkowitz, Trends Pharm Sciences28(8)416-422, 2007 and DeFea, Br J Pharm1-12, doi: 10.1038/sj. bjp.0707508, 2007, the authors concluded two independent and interdependent signaling pathways that start from activated 7TMR, which may involve both G and β arrestins; or it may involve only the G protein or only the beta-arrestin.

Thus, certain known antagonists, such as 7TMR, can activate β arrestin signaling. Propranolol (Propranolol) is a beta₂Known antagonists of adrenergic receptor (ADRB2) and G protein signaling; as observed in the practice of the present invention, it is part of a beta-arrestin signalingAgonists, capable of activating the pathway initiated by the beta-arrestin.

Cell signaling events in response to extracellular stimuli are generally mediated by protein-protein interactions. Thus, protein-protein interactions are of great importance to the cytophysiologist. One tool to monitor these interactions involves the use of a cleaved or sequence altered reporter activating protein, such as Tobacco Etch Virus (TEV) protease. When the interacting protein is co-expressed as a fusion construct, the cleavage portion of the protease regains activity. Wehr, et al, "Monitoring Regulated Protein-Protein Interactions using Split TEV," Nature Methods, 3: 985-993(2006). This property has been used in conjunction with transcription coupled reporter systems.

This understanding has led to other methods of determining activation and inhibition of GPCRs. One of the methods involves monitoring the interaction between a protein and an inhibitory protein in intact cells with a transcriptional machinery. The advantage of this approach is that knowledge of the G protein pathway need not be possessed. See U.S. patent No. 7,049,076 to Lee et al: "method for determining protein-protein interactions". Lee et al teach a reporter system that requires a transcriptionally coupled reporter system. According to Lee et al, a peptide transcription factor is cleaved from a first protein when the two proteins interact. The second protein is a transcription factor that activates the reporter. The transcription factor is transferred to the nucleus of the cell to effect transcription of a detectable reporter molecule, thereby effecting the function of the reporter molecule. Since this method relies on transcription, it cannot be used, for example, for platelets, artificial packages or units such as liposomes, cochlear bacteria, virus-like particles and viral particles.

Oakley, et al, Assay Drug dev. 2130(2002) and U.S. Pat. Nos. 5,891,646 and 6, 110,693 to Barak et al, "Methods Of analyzing receptor activity and structures use in Such Methods" describe assays in which the redistribution Of fluorescently labeled arrestin molecules in the cytoplasm to cell surface activated receptors is measured. These methods rely on high resolution imaging of cells to measure the repositioning of the suppressor protein and the activation of the reporter molecule. Those skilled in the art recognize that this is a tedious and time consuming procedure that can fail due to the affinity and interaction of the complementing enzyme fragments used, which may compete with the expected modulator induced interactions. Thus, a disadvantage of this method is that the enzymes will automatically reassociate independently of ligand binding, leading to false positive results. It would be desirable to have an assay that is simpler, more reliable, has fewer false positive results, and is more amenable to high throughput screening.

Various other U.S. patents have been issued and multiple patent applications filed in connection with these disclosures. For example, U.S. Pat. No. 6,528,271 to Bohn et al, "Inhibition Of β -Arrestin media activities and tendencies OptiIdreceptor-media Analgesia," describes several assays for screening analgesic drugs in which Inhibition Of β -Arrestin binding is measured. Published U.S. patent applications such as 2004/0002119, 2003/0157553, and 2003/0143626, and U.S. patent No. 6,884,870 describe assays involving different forms of GPCRs. U.S. patent No. 7,128,915 describes similar GPCR technology. The above-mentioned U.S. Pat. No. 7,049,076 mainly describes GPCR activity or screening methods, and shows the importance of GPCR research.

Accordingly, it is a feature of the present invention that the provision of a simple assay for monitoring and/or determining modulation of specific protein-protein interactions, such as receptor-mediated physiological effects, e.g., GPCR-mediated cellular responses, including but not limited to membrane bound proteins, including receptors in general, GPCRs being an important example, satisfactorily addresses the need in the art.

Summary of The Invention

The present invention provides methods for determining whether a test compound modulates a particular protein-protein interaction of interest. Protein-protein interactions are a common mechanism in biology whereby cells interact with their surroundings. This is an extracellular event, e.g., binding of a ligand to a receptor, capable of producing an internal response with or without internalization of the ligand. Internalization may involve two or more proteins with a portion on or off the membrane. Thus, the formation of dimers, heterodimers or multimers may produce an internal response. Intracellular protein-protein interactions may also occur in the signaling cascade. The general principles of the present invention apply to any type of protein-protein interaction. For example, the interaction may occur between two membrane bound proteins, between one membrane bound protein and one cytoplasmic protein, or between cytoplasmic proteins, etc. One embodiment describes the transfer of a cytoplasmic protein to another organelle such as the nucleus, where the reporter is activated to generate a signal. Cell-based assays are preferred, but cell-free systems, such as using cuttings, membrane fractions, nuclear fractions, and the like, may also be used. The invention also includes artificial packages or units containing one or more agents of interest, such as liposomes, virus-like particles, and the like. The present invention improves upon the Lee et al method discussed above because transcription is not required. Thus, results can be obtained more quickly, and results can be obtained from cell-based or cell-free systems. The following is a general description of some embodiments that are particularly preferred. These embodiments are for exemplary purposes only and are not intended to limit the scope of the invention described herein and defined by the claims.

One feature provided by the present invention includes contacting at least one test compound with the surface of a cell expressing a protein of interest. The ability of a test compound to modulate the activity of a protein of interest, such as a receptor protein, can be assessed. Expression of the protein of interest in the cell can be caused by transformation or transfection of a selected cell, e.g., an insect or mammalian cell line, with: (1) one or more nucleic acid molecules comprising: (a) a polynucleotide encoding a first protein of interest, and (b) a polynucleotide encoding a reporter activating protein having a cleavage site sensitive to a protease or to an active or activatable portion of a protease, and (2) one or more nucleic acid molecules comprising: (a) a polynucleotide encoding a second protein whose interaction with the first protein of interest is altered in the presence of a modulator, such as a positive test compound, and (b) a polynucleotide encoding a protease or an active or activatable portion of a protease specific for the cleavage site encoded by nucleic acid (1). The assessment or analysis of molecules that modulate the interaction (between the two proteins studied), such as a positive test compound, can be performed by adding a reporter activating protein substrate, if necessary, to the cells expressing the first and second proteins studied, as well as to the reporter system described herein.

Thus, one method of the invention may be to employ a re-orderable enzyme as a readout for the protein-protein interaction under investigation. The reorderable activating protein, e.g., enzyme, used as a reporter or reporter activating protein may be in an inactive state and may be activated by cleavage, e.g., by enzymatic activity associated with the second protein of interest. Another option includes an inactive reporter activating protein that is activated when the first and second proteins of interest interact. In this way, compounds that modulate the interaction between the first and second proteins of interest can be screened. One component of this system is the ability to identify molecules capable of modulating selected protein-protein interactions at high throughput.

The enzyme capable of producing the readout "peptide a" (alone or together with one or more related molecules) is present in a form in which the activity can be varied. This change in activity can activate or inactivate the enzyme. For example, a cleavage site may be created in the enzyme such that it is inactivated upon cleavage, e.g., it may be cleaved by a second enzyme coupled to a second protein of interest.

Alternatively, the cutting may also result in activation. The selected enzyme may be constructed into the desired host cell using one or more nucleic acids. For example, a vector may comprise a polynucleotide encoding a selected molecule as an inactive enzyme that can be activated by cleavage of the inactive enzyme at a cleavage site. The cleavage site may occur naturally, but more preferably the cleavage site is incorporated into the polynucleotide such that it is expressed as a rearranged enzyme. For example, a cleavage site not otherwise associated with the cellular protein and/or a protein not otherwise associated with the host cell may be transfected into the host cell. Alternative embodiments include activation of the enzyme by cleavage, either by removal of a blocking peptide, or by allowing the two polypeptides to change configuration and rearrange them to activate the enzyme activity.

Accordingly, one embodiment features an active polypeptide, e.g., an enzyme. The "enzyme" described herein can be inactivated by cleavage. From a specificity standpoint, it may be desirable to construct a cleavage site in the enzyme that is recognized by a protease not otherwise associated with the host cell. The cleavage site may be introduced as a linker joining two parts of the "enzyme" together by a linking component, the linker may be a cleavage site originally belonging to the "enzyme", e.g., the enzyme with the cleavage site may be from another cell type or another species and not present in the host cell, or the cleavage site may be generated by conservative substitution of one or more amino acids. Conservative substitutions are known in the art. For example, charge, size, aromaticity, or other properties may be retained to maintain activity. This activity need not be identical to that of an "enzyme" whose sequence is not rearranged, but must be altered by cleavage at the cleavage site. A cleavage site may be inserted between two parts of an enzyme. Cleavage of this site may interfere with the enzyme, leading to inactivation or possibly causing catalytic activity, for example, by removing portions of the peptide that block the binding site or catalytic site or allowing two portions of the rearranged enzyme to interact in a manner that restores activity.

Thus, cleavage at the cleavage site may inactivate or activate the protein that produced the readout. Cleavage can be effected in the presence of a test compound, for example, when the expression product of a nucleic acid molecule comprising a polynucleotide encoding a second protein of interest interacts with the first protein of interest, thereby triggering the activity of a protease that recognizes and cleaves a protease-sensitive cleavage sequence in the rearranged reporter-activated protein.

The second protein of interest interacts with the first protein of interest in the presence or absence of the third molecule. This third molecule is therefore believed to modulate the protein-protein interaction between polypeptides a and B. Thus, protein-protein or peptide-peptide interactions (for purposes of discussion, proteins and peptides may be used interchangeably) modulated by a third molecule, such as a test compound, may be effectively reported by the system of the present invention. Molecules that modulate protein-protein interactions (between polypeptides labeled 1 and 2, first and second, A and B, etc., these phrases and terms are used interchangeably herein) can be measured by an active reporter activating molecule, or by adding a substrate for an active reporter activating protein to a cell expressing a system containing the protein of interest.

The choice of proteins a and B is a design choice, as pairs of molecules that are known or suspected to be likely to associate, interact with each other may be used. As discussed herein, one suitable molecular pair is 7TMR with a G protein or a β arrestin. Another example is a frizzled receptor and a random binding protein, etc. Yet another example is a protein that functions during and after cellular interactions. Thus, proteins a and B are in cell 1. When the cell 1 is contacted by the cell 2 or contacted with the cell 2, this interaction triggers an activity in the cell 1 caused by the cell 1, as revealed by the association, interaction, etc. of the proteins a and B, and further as revealed by the agents of the invention, produces a detectable and detectable signal.

Another example is to express protein A on cell 1 and protein B on cell 2. This can be accomplished by constructing the G protein or the beta-arrestin to have an extracellular domain that is exposed to cell 1 upon activation of cell 1 by a ligand or candidate drug, or by constructing the reporter activating protein to have an extracellular domain. Alternatively, endogenous molecules on both cells may also associate spontaneously. In another embodiment, the protease and reporter activating molecule are configured to be expressed as an extracellular domain on the surface of a cell or unit.

In another embodiment, the interrelated, assembled proteins form a composite structure comprising proteins a and B. The assay can be used to identify molecules that promote or prevent association or assembly. One example is the formation of viral capsids, assembly of virus-like particles or the formation of ribosomes.

In the common mechanism of G protein-coupled receptors (GPCRs are also known as 7TMR, these terms being used interchangeably herein), agonist activation of GPCRs results in the recruitment of an intracellular molecule, such as G protein or β arrestin, that is involved in a signaling pathway, such as initiation, termination, enhancement, resistance, and the like. Thus, G protein-coupled receptor kinases can act on activated receptors, leading to phosphorylation of the receptors. Phosphorylated receptors promote the binding of beta-arrestin to the receptor. For some GPCRs, this mechanism is quite conservative. In other cases, the activated receptor interacts with a beta arrestin.

To evaluate molecules that modulate protein-protein interactions such as GPCR activation, a system was designed to measure protein-protein interactions and tested in a GPCR-rearranged reporter system. For example, the reporter system may be a luciferase/luciferin assay system. Generally, a reporter is an exogenous molecule, i.e., foreign to the host cell or signaling mechanism. This minimizes the extent to which the reporter is activated by or spontaneously in the host cell, thereby also minimizing signal generation and thus false positive results. The reporter activating molecule may be a molecule with a domain structure or a molecule whose sequence is rearranged to produce an inactive reporter activating protein which may have reporter activity when manipulated. Thus, the present application contemplates the use of a potential reporter activating molecule. The rearranged reporter activating molecule minimizes the activity of the spontaneous reporter activating molecule and thus minimizes false positive results. For example, in enzyme fragment complementation assays, the affinity of the enzyme fragment may exceed the reaction kinetics of the target molecule, ligand or molecule being screened, allowing the enzyme fragment to spontaneously reassociate into functional molecules, resulting in higher background values and/or false positives. The rearranged reporter activating protein of interest can be constructed with a site that, when acted upon, causes the rearranged reporter activating molecule to form a functional molecule. This site may be a protease site. The protease site is preferably a unique site that is rarely or not present in the host cell, or a unit in which one or more components of the method of interest are present. This provides another means to avoid spontaneous reassociation of intact reporter activating molecules, thereby minimizing false positive results. A specific signal is only obtained when the conjugated ligand eventually directs the protease to the vicinity of the inactive reporter activating protein and causes it to cleave, and only then is an active signal activating or generating entity achieved. Many proteases are known in the art to be useful in the practice of the present invention. For example, proteases of viral origin may be useful because they are usually exogenous to the intact host cell. One application involves the use of a rearranged reporter-activated protein gene in which the coding sequence for firefly luciferase is tagged at the C-terminal end of the GPCR sequence, and β -arrestin 2(Ar2 or Arr2) is linked to the Tobacco Etch Virus (TEV) protease gene. In another embodiment, the rearranged luciferase is tagged with β arrestin (Ar or Arr) and the TEV gene is linked to a protein that is affected by β arrestin or downstream of the signaling pathway associated with β arrestin, or to a receptor, such as 7TMR, suspected of acting independently of the G protein. When plasmids designed to express both are expressed in cells, compounds that modulate GPCR-arrestin-2 interactions recruit Arr 2-protease fusion proteins to the protease recognition site in the permuted luciferase, which is then cleaved by TEV protease. The utility of a test compound can be measured by the change in enzyme activity resulting from reconstitution of the reporter-activated protein, in which case the luciferase becomes active and can produce a detectable signal by acting on an appropriate substrate such as luciferin.

The invention is not limited to luciferases, or even to enzymes. Activation by cleavage is a known phenomenon, such as zymogen. Non-enzymatic reporter systems may also be used. For example, Green Fluorescent Protein (GFP) can be used. A rearranged GFP, e.g., a rearranged GFP component thereof, is useful as a reporter activating protein and reporter. The action of a protease comprised by the rearranged polypeptide, such as TEV or other protease with a recognition site, cleaves the rearranged reporter-activating protein/reporter, thereby allowing signal rearrangement. GFP has the advantage of being a detectable reporter signaling molecule in its own right. Alternatively, cleavage sites may be introduced into the reporter that do not significantly interfere with the signal. Cleavage by protein-protein interactions results in a reduction in reporter signal. Multiple cleavage sites can be introduced into the reporter construct.

The tertiary structure of the protein can be used to guide the skilled artisan to place the cleavage site in the most appropriate position. For example, when two portions of a polypeptide are brought into close contact, separating the two portions in a manner that perturbs the sequence would be expected to reduce or eliminate activity. When cleaved, the two moieties are expected to interact, thereby restoring activity.

The first protein of interest may be a membrane bound protein such as a transmembrane receptor, for example a GPCR. Examples of transmembrane receptors include β -adrenergic receptor (ADRB2), arginine vasopressin receptor 2(AVPR2 or V2), serotonin receptor 1a (HTR1A), m2 muscarinic acetylcholine receptor (CHRM2), chemokine (C-C motif) receptor 5(CCR5), dopamine D2 receptor (DRD2), kappa-opioid receptor (OPRK), or α 1 a-adrenergic receptor (ADRA1A), and the like. Membrane bound receptors are well known in the art. It is to be understood that the invention is not limited in all respects to the specific embodiments described as examples of the invention. For example, molecules such as the insulin growth factor-1 receptor (IGF-1R), a tyrosine kinase, and proteins that are not normally membrane bound, such as estrogen receptor 1(ESR1) and estrogen receptor 2(ESR2), may be used in the present invention. The protease or a portion of the protease associated with protein B may be tobacco etch virus nuclear inclusion a (tev) protease. TEV has a seven residue recognition site and is therefore more specific than proteases with smaller, statistically more common recognition sites. Other proteases are also suitable for use in the present invention. For example, enterokinase and factor Xa proteases with a 5 residue recognition sequence, thrombin and Pureact with a 6 residue recognition sequence^TMOr CleanCut^TMAnd PreScission having a 7-residue recognition sequence^TMAre also proteases that can be used in the present invention. The present invention is not limited to the use of any particular protease. However, the protease must cleave at a certain site, thereby generating or altering a signal from the receptor.

The protein that activates the reporter can be any enzyme that acts on the substrate to produce a detectable signal. For example, the enzyme may increase or decrease fluorescence or chemiluminescence, directly or indirectly, or may cause a color change. The reporter substrate may be a biological substance, such as a protein, or may be a chemical substance whose reaction is catalyzed by a reporter enzyme. The second protein of interest may be an inhibitory protein, such as an arrestin. Profilins typically interact with GPCRs in response to ligand/receptor interactions to modulate activity. The cell may be a eukaryotic cell or a prokaryotic cell. The reporter molecule may be an exogenous component, such as β -galactosidase or luciferase. For simplicity, "reporter enzyme" is used as a reporter activator, reporter modulator protein, or equivalent of a reporter activating protein, and is used as a shorthand for a molecule that alters the output of a reporter. For example, the reporter enzyme may cause a change in the reporter signal enzymatically, or may cause a change in the signal enzymatically or non-enzymatically, such as a change in a fluorescent signal. Those skilled in the art are aware of various reporter systems and proteins that modulate or activate reporter signaling.

The nucleotide sequence encoding the first protein may be modified to increase interaction with the second protein. Such modifications include, but are not limited to, the replacement of all or part of the nucleotide sequence of the C-terminal region of the first protein with a nucleotide sequence encoding an amino acid sequence having a higher affinity for the second protein than the original sequence. For example, the C-terminal region may be replaced by a nucleotide sequence encoding the C-terminal region of AVPR2, AGTRLI, F2RL1, CXCR2/IL-8b or CCR 4. Such modifications are well known in the art and are an optional feature of the present invention.

The methods of the invention may comprise contacting a plurality of test compounds with a plurality of cell samples or units. Each sample may be contacted with one or more test compounds. In another embodiment, one cell or unit carries two different molecules, and the extracellular domains of these molecules carry different reporter activating molecules, both of which interact with the β -arrestin. The screening process can be accomplished by measuring the activity of the reporter, for example by monitoring the activity of the enzyme in the sample to determine if any compound or mixture of compounds modulates a particular protein-protein interaction. The method can include contacting each sample with a single test compound, contacting each test sample with a mixture of test compounds, or a combination of both. The present invention can be used to test or screen for compounds that inhibit binding between the compound and protein a. For example, an assay may include a known ligand for protein A, and compounds that modulate binding between the ligand and the protein may also be identified and/or identified, as in a competition assay. Control samples can be used for each analysis, or in parallel with the sample.

In certain embodiments, the invention provides a method for determining whether a test compound modulates one or more interactions between a plurality of proteins of interest. These embodiments generally have the following features: contacting a test compound with a sample of cells transformed or transfected with: (a) a first nucleic acid molecule comprising: (i) a polynucleotide encoding a first protein and a polynucleotide sequence encoding a protease cleavage site, (ii) a polynucleotide encoding a protein that activates a reporter in a cell; and (b) a second nucleic acid molecule comprising: (i) a polynucleotide encoding a second protein whose interaction with the first protein is to be measured in the presence of the test compound under investigation, (ii) a polynucleotide encoding a protease or encoding a polypeptide specific for cleavage by a cleavage site. The first protein may be different from the other first proteins in the plurality of samples. The method then includes determining the activity of the reporter in one or more of the plurality of samples, thereby determining modulation of one or more interactions between the proteins of interest.

The second protein may be different or the same in each sample. All samples may be combined in a common container, and each sample may comprise a different pair of first and second proteins. Alternatively, each sample may be tested in a different container. The reporter in a given sample may be different from the reporter in other samples. The mixture of test compounds may comprise or be present in a biological sample, such as cerebrospinal fluid, urine, blood, serum, pus, ascites, synovial fluid, tissue extracts, plant or herbal extracts, or exudate.

In other embodiments, the invention provides a recombinant cell transformed or transfected with: (a) a nucleic acid molecule comprising the following components: (i) a polynucleotide encoding a first protein, (ii) a polynucleotide encoding a protease, a portion of a protease, or a cleavage site for a polypeptide having protease activity, and (iii) a polynucleotide encoding a protein that activates a reporter in a cell; and (b) a nucleic acid molecule comprising: (i) a polynucleotide encoding a second protein whose interaction with the first protein is to be measured in the presence of the test compound under investigation, and (ii) a polynucleotide encoding a protease, a portion of a protease, or a polypeptide having protease activity specific for the cleavage site.

One or both of the nucleic acid molecules may be stably integrated into the genome of the host test cell. The cell may also be transformed or transfected with a reporter molecule. The first protein may be a membrane bound protein, such as a transmembrane receptor, for example a GPCR. Representative transmembrane receptors include ADRB2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK, or ADRA 1A.

As mentioned above, the protease or a portion thereof is not limited to tobacco etch virus nuclear inclusion protein A enzyme, but can be any protein that activates a reporter activating protein, and can be any enzyme that acts on a substrate to produce a useful or detectable signal. The second protein may be an inhibitory protein. The cell may be a eukaryotic cell or a prokaryotic cell, and in general, eukaryotic cells are preferred for the purpose of screening drugs. Cells that are glycosylated in a manner similar to the ultimate target of the drug are particularly preferred. The cells may be cultured or engineered to provide the desired glycosylation characteristics. The use of prokaryotic cells that do not match the glycosylation profile of the proposed end target may be useful for screening and identification.

The reporter molecule may be exogenous, for example β galactosidase, GFP or luciferase. The nucleotide sequence encoding the first protein may be modified to increase interaction with the second protein, for example by replacing all or part of the nucleotide sequence of the C-terminal region of the first protein with a nucleotide sequence encoding an amino acid sequence having a higher affinity for the second protein than the original sequence. The C-terminal region may be replaced by a nucleotide sequence encoding the C-terminal region of, for example, AVPR2, AGTRLI, F2RL1, CXCR2/IL-8b or CCR 4.

As one embodiment, the invention includes providing an isolated nucleic acid molecule comprising (i) a polynucleotide encoding a protein, (ii) a polynucleotide encoding a protease, a portion of a protease, or a cleavage site for a polypeptide having protease activity, and (iii) a polynucleotide encoding a protein that activates a reporter in a cell or other assay system. The protein may be a membrane-bound protein such as a transmembrane receptor, for example a GPCR. Representative transmembrane receptors include ADRB2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK, or ADRA 1A. The protease or a portion of the protease may be a tobacco etch virus nuclear inclusion protein a enzyme. As mentioned above, the protein that activates the reporter can be any protein that reacts with a substrate to produce a signal and need not be limited to the TEV examples discussed herein. This and other examples of the invention are not to be considered as limiting the invention to the particular embodiments.

In certain embodiments, the invention features an expression vector comprising an isolated nucleic acid molecule comprising: (i) a polynucleotide encoding a protein, (ii) a polynucleotide encoding a protease, a portion of a protease, or a cleavage site for a polypeptide having protease activity, and (iii) a polynucleotide encoding a protein capable of activating a reporter molecule in a cell, further operably linked to a promoter.

In certain embodiments, the invention features an isolated nucleic acid molecule comprising: (i) a polynucleotide encoding a protein whose interaction with another protein is to be measured in the presence of a test compound, and (ii) a polynucleotide encoding a protease, a portion of a protease, or a polypeptide specific for the cleavage site. The protein or other protein may be an inhibitory protein, such as an inhibitory protein.

Certain embodiments of the invention also feature an expression vector comprising an isolated nucleic acid molecule comprising: (i) a polynucleotide encoding a protein whose interaction with another protein is to be measured in the presence of a test compound, and (ii) a polynucleotide encoding a protease, a portion of a protease, specific for the cleavage site, and the nucleic acid may further be operably linked to a promoter.

Another embodiment features a fusion protein produced by expression of an isolated nucleic acid molecule comprising: (i) a polynucleotide encoding a protein, (ii) a polynucleotide encoding a protease, a portion of a protease, or a cleavage site for a polypeptide having protease activity, and (iii) a polynucleotide encoding a protein that activates a reporter molecule in a cell, further operably linked to a promoter; or an isolated nucleic acid molecule comprising: (i) a polynucleotide encoding a protein whose interaction with another protein is to be measured in the presence of the test compound under investigation, and (ii) a polynucleotide encoding a protease, a portion of a protease, specific for said cleavage site.

In other embodiments, the invention features kits for determining whether a test compound modulates a particular protein-protein interaction of interest. The kit comprises one or more of the following components, wherein the components are separated from each other: (a) a nucleic acid molecule comprising a polynucleotide encoding a first protein, (i) a polynucleotide encoding a protease, a portion of a protease, or a cleavage site for a polypeptide having protease activity, (ii) a polynucleotide encoding a protein that activates a reporter molecule in a cell, and (b) a nucleic acid molecule comprising: (i) a polynucleotide encoding a second protein whose interaction with the first protein in the presence of the test compound is to be measured, (ii) a polynucleotide encoding a protease or a portion of a protease specific for said cleavage site; and optionally, means for maintaining (a) and (b) separate. The kit may also include instructions for use. Alternatively, the kit may also include cells engineered to express either or both of the two fusion proteins of interest.

The first protein may be a membrane-bound protein, such as a transmembrane receptor. One particular type of transmembrane receptor is the GPCR. One particular transmembrane protein is a GPCR. Representative transmembrane receptors include ADRB2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK, or ADRA 1A. The protease, a portion of the protease, or a polypeptide having protease activity can be a tobacco etch virus nuclear inclusion protein a enzyme. The protein that activates the reporter can be, for example, any protease that acts on a detectable reporter activating molecule that is responsive to cleavage activation. The reporter molecule can be any molecule that produces a detectable signal from the cleavage product. The second protein may be an inhibitory protein, such as an arrestin. The kit may further comprise a separate isolated nucleic acid molecule encoding a reporter activating gene. The reporter activator may for example be a beta galactosidase or luciferase. The nucleotide sequence encoding the first protein may be modified to increase its interaction with the second protein, for example by replacing all or part of the nucleotide sequence of the C-terminal region of the first protein with a nucleotide sequence encoding an amino acid sequence having a higher affinity for the second protein than the original sequence. The nucleotide sequence of the C-terminal region may be replaced by nucleotide sequences encoding the C-terminal region of, for example, AVPR2, AGTRLI, F2RL1, CXCR2/IL-8B and CCR 4.

It is contemplated that any method or composition described herein can be practiced with respect to any other method or composition described herein. In the claims and/or the specification, the articles "a" and "an" when used in conjunction with the word "comprising" may mean "an" but may also mean "one or more", "at least one", and "one or more". When corresponding components are described in slightly different words, they are not meant to distinguish between the embodiments, and the words broadly describe the corresponding components as a whole.

These and other embodiments of the present invention will be better understood when considered in conjunction with the following description and the accompanying drawings. It should be understood that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

Brief description of the drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, further illustrate features of certain aspects of the invention. The invention will be better understood upon reading the detailed description provided herein in connection with a particular embodiment, and by reference to one or more of these drawings appended hereto.

FIG. 1 shows a schematic of an embodiment of the invention in which modulator 4 binds to a protease-linked protein 1, resulting in the protein interacting with a second protein 2 linked to a reporter modulator 3, such as a rearranged inactive regulatory protein. Modulator 4 represents a compound that modulates protein-protein interactions. In this example, Ar or arrestin 2 is fused to an inactive rearranged reporter regulatory or activating protein 3. Protease 7 is linked to protease 1. One cleavage site 8 is shown between two fragments of reporter regulatory protein 3. When protease cleavage occurs following interaction with a modulator 4 attached to a protein 1, such as 7TMR, the reporter-activated protein activity is reconstituted, ultimately producing a detectable signal as represented by light bulb 6.

FIG. 2 is a diagram of a protein-protein interaction assay method of the present invention. A membrane-bound protein 21 with intracellular protease 22 interacts with modulator 4. An inactive reporter linked to protein 23 carries an inactive reporter 3 or reporter activator 3. Upon interaction, the protease 22 associated with protein 21 cleaves the rearranged reporter activating protein 23 at the cleavage site, thereby rearranging the protein portion of reporter activating protein 3 to 5 upon engagement of modulator 4 with protein 21. The reporter activator thereby effects recombination of the reporter or reporter activator 3 to cause or activate a reporter.

FIG. 3 shows a schematic diagram of an embodiment in which two transmembrane proteins interact. Molecule 4 causes (modulates) the interaction between two membrane proteins, e.g. at least one receptor protein. In the figure, a protease 22, such as TEV (7 in FIG. 1), is linked to protein 1 and brought into proximity of the rearranged reporter activator fusion protein 3, which is linked to a second membrane protein 33. Proteolysis at cleavage site 8 due to proximity of proteins 1, 33 results in activation 5 of reporter activated protein 3.

FIG. 3 can also be viewed as a scheme showing the application of an identification technique to molecules that modulate the formation of reporter homodimers or heterodimers. In the figure, proteins 1 and 33 are membrane-bound proteins. Proteins 1 and 33 are constructed to each comprise a protease 22 or a reporter 3 activated by protease 22. The molecule that modulates the interaction 4 binds to protein 1 and/or 33. When 1 and 33 interact, protease 22 acts on reporter activator 3, thereby generating an altered signal. Heterodimerization may be extended to include conventional homodimerization. For example, 1 and 33 may be two copies of the same reporter, but differ in binding to the protease or reporter. As discussed elsewhere, the terms reporter and reporter activator are often used interchangeably to describe various embodiments of the invention.

FIG. 4 shows an example of protein-protein interactions involving intracellular proteins. Protein 41 is a nuclear hormone receptor fused to an activator 22 such as TEV. A rearranged reporter activating molecule 43 is located in the nucleus 40. The reporter molecule can be localized in the nucleus of the cell by using a basic polypeptide having the function of a nuclear localization peptide sequence. Upon binding to a ligand such as a hormone (not shown here), the NHR fusion 41 translocates to the nucleus where it interacts with a reporter system.

Figure 5 shows that the luciferase activity regenerated in the permuted luciferase fusion protein by TEV protease in the cell can be controlled by modifying the cleavage site of the protease. The signal to noise ratio can be controlled. Two constructs are shown here, ADRB2 being the β 2 adrenergic receptor, Luc234-550 and 2-233 being two fragments of luciferase joined by X (i.e., a TEV cleavage site with a variable C-terminal amino acid). For example, X may be serine S, arginine R, or valine V. When both constructs were present in one cell, reconstituted luciferase activity was observed in mammalian cells from the rearranged luciferase fusion protein. The sensitivity to TEV cleavage depends on the specific residue at the cleavage site. RLUs here and elsewhere denote relative luminescence (or light) units. No ligand was used in this experiment.

FIG. 6 shows agonist-induced luciferase activity in a GPCR-permuted luciferase cell-based assay using ADBR2 receptor linked to a permuted luciferase with a different TEV protease cleavage site, R at position X of the TEV cleavage site (left panel) and V at position X (right panel). TEV protease is fused to an inhibitor protein. The x-axis of each figure shows zero values (no agonist) and 10 μ M agonist.

FIG. 7 shows dose-dependent response of luciferase activity in GPCR-rearrangement-based luciferase cell assays in co-transient and partially transient transfection systems. In the left panel, valine of the protease cleavage site construct was used in CHO cells. Both ADRB2-luc and Arr-TEV constructs were transiently co-transfected. In the right panel, cells were stably transfected with the luciferase construct fused to ADRB2 and containing R, and then transiently transfected with the Arr-TEV construct.

FIG. 8 shows an alternative GPCR-rearranged luciferase assay. The expression construct carries ADRB2 bound to a reporter activator, and arrestin 2 bound to a protease. These constructs were transiently transfected into HEK293 cells. The kinetics of the reaction are shown in the figure as 1 hour (. tangle-solidup.) and 5 hours (■) of the reaction culture.

FIG. 9 shows the generation of a HEK cell line stably expressing the arrestin/rearranged enzyme construct carrying v at position X and transiently transfected by the receptor ADRB2-TEV (left panel), and a CHO cell line stably expressing Arr-luc234S233 and transiently transfected by the receptor-TEV construct.

FIG. 10 shows the results for agonist (isoproterenol) (■), partial agonist (. tangle-solidup.), antagonist plus isoproterenol in HEK cells stably expressing the profilin/rearranged enzyme construct and transiently transfected with ADRB2-TEVAntagonists (. diamond.) and a Per-Luc assay for non-specific endogenous receptor (●) response.

FIG. 11 shows GPCR Per-Luc assay evaluation of V2 (vasopressin receptor 2) agonist and inverse agonist. HEK cells stably transfected with the profilin/rearranged luciferase construct were transiently transfected with the V2-TEV construct and induced by agonist, 8AVP, arginine vasopressin (left panel). When these cells were analyzed with inverse agonists, a dose-dependence was observed, with the signal being mediated by the arrestin, but not by the G protein (right panel).

Figure 12 shows several beta arrestin-based assays for ADRB 2. In the right panel, discoverrx HEK cells were transiently transfected with ADRB2 as per manufacturer's instructions and tested with one antagonist (propranolol) (■), agonist (isoproterenol) (tangle-solidup) and a combination of both (●) (left) and on the right with agonist (■), inverse agonist (tangle-solidup) and a combination of both (●). The results of the right hand side assay are compared to the results of the assay of the invention for response to the antagonist, isoproterenol agonist, and a combination of the two. The assays of the invention on the left provide greater discriminatory power and higher specific activity.

Figure 13 shows a β arrestin-based assay of V2. In the figure, the V2 inverse agonist (SR121463) induced a signal that was independent of the G protein and dependent on the arrestin.

FIG. 14 shows several expression constructs comprising an overlap of substantially full-length, but not complete, copies of luciferase enzyme linked to form a permuted luciferase according to the methods taught herein. CMV is a cytomegalovirus promoter. Luc2-456 and Luc234-550 are essentially full-length luciferase fragments. In this example, GS is a peptide linker consisting of glycine and serine. The TEV cleavage site has a valine at the C-terminus.

FIG. 15 shows an example of an assay for monitoring intracellular protein-protein interactions. Rapamycin (Rapamycin) is an immunosuppressive drug that binds both to a Rapamycin-binding protein (FKBP12 or FKBP) and to the FKBP-Rapamycin (FRB) binding domain of a Rapamycin mammalian target (mTOR) kinase. mTOR is a murine serine/threonine protein kinase that contains the rapamycin binding domain 151, with rapamycin binding domain 151 being the mammalian target for rapamycin 154. FKBP152 is a 12kDa FK 506-binding protein with a rapamycin binding site. TEV protease is fused to the rapamycin-binding domain of mTOR, FRB 151. The rearranged reporter activating protein is then fused to the rapamycin binding domain of FKBP152, FKBP 12. Rapamycin 154 reacts with, mediates binding to, and brings them into proximity with FRB151 and FKBP152, leading to activation of the rearranged reporter.

Figure 16 shows the configuration of an assay in which proteins a21 and B23 are two membrane bound receptors that spontaneously dimerize (left to right) or dissociate (right to left) upon binding to a ligand. The assay can monitor spontaneous or induced interactions between two receptors, where one or both receptors bind to the same or different ligands. Alternatively, proteins a21 and B23 may dimerize spontaneously or without binding to a ligand or modulator (not shown). In this embodiment, the rearranged reporter activator moieties of the protease and fusion protein are expressed on the cell surface or on the exterior of the artificial package or unit. The assay may also be configured to monitor for disruption of the interacting receptor, as indicated by attenuation, reduction or disappearance of the signal, whether such disruption is spontaneous or mediated by one or more molecules.

FIG. 17 shows a cellular assay in which proteins A171 and B172 are present in different cells that may bind, abut, interact, or the like. Similarly, a171 or B172 may carry protease 177 or rearranged signal activating protein 173. The assay can detect spontaneous or induced interactions between two labeled receptors on two cells as evidence of cell-cell interaction or proximity, where one or both receptors bind to a single ligand, which may be the same or different. In this embodiment, the rearranged reporter activator portion 173 of protease 177 and fusion protein is expressed on the cell surface or on the exterior of the artificial package. Activated rearranged reporter 175 results from the binding of proteins a171 and B173 to each other. Alternatively, the receptor fusion and intracellular protein fusion of interest may be present in the same cell, and the induction event, ligand, etc. being monitored is expressed on or is the second cell. The assay may also be configured to monitor for disruption of the interacting receptor and cell, such as that indicated by attenuation, reduction or disappearance of the signal as the cell separates, whether such disruption is spontaneous or mediated by one or more molecules.

Detailed description of some representative embodiments

The assay of the invention detects protein-protein interactions without prior knowledge of the compounds that modulate the interaction or the cellular pathways that result from the interaction. The assay can detect interactions between membrane proteins, such as the formation of dimers or heterodimers. The assay can detect interactions between membrane proteins and cytoplasmic proteins. This assay can detect the interaction of two cytoplasmic proteins. The assay detects translocation of a protein to intracellular space or intracellular organelles. The assay can detect an interaction between two cells or packages or units. Protein A or B may bind to a ligand, cofactor, or other compound, molecule, or substance that may not be essential for protein-protein interactions.

As known to those skilled in the art, the term "sequence" has a variety of uses in the fields of genetic engineering, nucleic acids, and proteins, and may have different meanings in the context of a sentence, paragraph, concept, idea, section of text, and the like. For example, a sequence may represent a particular list of amino acid residues (primary structure) of a polypeptide, or a list of nucleotide bases of a polynucleotide. In another context, a sequence may refer to a broad range of complex molecules, such as a polypeptide sequence that refers to the entire molecule without knowledge of the primary amino acid structure. A gene sequence may be synonymous with a gene and refers to the polynucleotide itself or in its entirety. Sequence may refer to a single polypeptide or polynucleotide, or to a portion thereof. Thus, when the phrase "sequence operably linked" is used, it is meant that individual genes, domains or transcriptional units may be linked or associated together in a functional fashion such that the associated or linked individual genes, domains, transcriptional units, etc. are expressed. These sequences may also be part of a particular expressed gene or protein, such as a domain of a protein having multiple functional portions or domains. The polynucleotide of interest may be DNA or RNA, or a mixture of both, as is known in the art. Methods of making and using these polynucleotides in the practice of the present invention are known in the art.

For example, FIG. 4 shows an assay for nuclear hormone receptor activity after modulation. Due to the rearrangement of the reporter activating protein and optionally a molecule that is detectable and can act as a reporter, the transport of the nuclear hormone receptor into the nucleus causes or provokes a signal, e.g. a change in fluorescence or the presence of fluorescence, which occurs in response to the activity of the activating protein, e.g. luciferase. The generated signal may be any detectable change, such as a change in intensity or a change in excitation/emission parameters. Chemiluminescence is another common reporter signal. One skilled in the art will appreciate that a protease or a rearranged reporter can be constructed having nuclei and other polypeptides of interest. Such target polypeptides may comprise basic amino acids. When the two interact in the target area, the signal is adjusted.

For GPCRs, the assay is specific, sensitive, and does not require prior knowledge of the specific G protein coupling. This assay is independent of endogenous GPCRs and can be used to identify molecules, including agonists, antagonists and inverse agonists of (certain receptors). The assay of the present invention is an improvement over the Lee et al method, avoiding the need for transcription amplification. The present invention provides a more rapid and direct reading.

The present invention provides a simpler, more reliable assay system than Lee et al, in part because the system does not require transfer of reagents to the nucleus and subsequent transcription of amplified signals. Thus, the reading can be approximated for receptor modulating events. Unlike the Lee et al method, the present invention requires nuclei. In fact, one application of the present invention is the detection of secreted proteins. Both the reporter molecule and the protease in the cytoplasm can activate (or inactivate) the signal from the secreted chaperone protein. Another embodiment is for an enucleated cell, or an artificial cell, package or unit.

The invention is particularly useful for identifying molecules that modulate any protein-protein interaction. Discoverx^TMAnalysis ofIs an assay using a beta-arrestin that requires that two interacting protein components be held together at all times to generate a signal. Most previous GPCR assays are based on G protein signaling, such as FLIPR and cAMP assays. Any influence of Ca⁺⁺Or cAMP levels are prone to false positive signals. On the other hand, the method of the invention is fast, reliable and cost-effective, while being independent of enzyme component binding or G-protein signaling that may affect sensitivity and specificity.

The present invention provides a means to analyze or screen for any protein-protein interaction by fusing protein A (or protein B) to a reporter activating protein to which the reporter modulating protein/molecule or reporter activating protein/molecule is equivalent. An example of this approach is an enzyme containing a rearrangement of proteolytic cleavage sites. Protein B is fused to a protease. The interaction of protein a and protein B may be constitutive or induced by a third molecule. One skilled in the art can use the assays of the invention to identify molecules that enhance or interfere with protein-protein interactions. Alternatively, protein A may be fused to a protease and protein B may be fused to a reporter activating protein.

The reporter activating protein of the present invention is latent and can be activated upon interaction with a protease of a second protein. One interesting approach is to generate a reporter activating protein, which is a rearranged molecule, constructed to contain protease cleavage sites. When cleaved, portions of the reporter activating protein can bind, assemble, and the like to produce an active reporter activating polypeptide or assembly. This active, e.g., enzymatically active reporter activating protein can then act on an appropriate substrate, e.g., a reporter, to generate a detectable signal. For example, where the rearranged, inactive molecule is a luciferase, when cleaved to form a biologically active luciferase, the luciferase may act on a suitable substrate, such as luciferin, to generate a detectable signal, in this case luminescence.

In another embodiment, the reporter activating protein is a reporter. This can therefore be seen as a self-activation of the reporter activating protein upon cleavage. An example is GFP, which rearranges upon cleavage and produces a detectable signal independent of the reporter system; for example, when the reporter activator is luciferase, the reporter molecule is a reagent that provides luciferin.

The rearranged reporter activating gene can be constructed in an active or inactive form. For example, at the time of developing this technology, a GPCR-inactive permuted luciferase fusion was constructed in which the order of the luciferase amino acid sequence was changed. The original N-terminal fragment was moved to the C-terminus, the original C-terminal fragment was moved to the N-terminus, and a protease recognition site was used to fuse the two permuted fragments. Interaction between a GPCR-inactive permuted luciferase fusion protein and a β arrestin 2-TEV protease fusion protein results in cleavage of the inactive permuted luciferase and the generation of a reconstituted luciferase activity. Using an alternative strategy, the inventors constructed a GPCR-active permuted luciferase fusion construct in which a protease recognition site was introduced into the original sequence of luciferase without significantly affecting luciferase activity. Interaction between GPCR-active permuted luciferase fusion protein and β arrestin 2-TEV protease fusion protein results in cleavage of the active permuted luciferase and the generation of two inactive luciferase fragments, resulting in loss of activity and thus diminished or lost signal.

Reporter activating proteins can be selected from reporters based on rearranged proteins, such as Gaussia luciferase; renilla luciferase (renilla luciferase); a beta lactamase; beta-galactosidase; and fluorescent proteins, such as Green Fluorescent Protein (GFP) or DsRed protein, etc., that contain proteolytic cleavage sites, such as TEV cleavage sites. Although "enzyme" is used as a generic term, the reporter activating protein itself is not limited to "enzyme", but refers to any reporter activating protein that is capable of producing a change in signal. For example, binding or chelating a fluorescent protein may produce a sufficient signal change without the need for chemical reactions that alter the molecular structure.

As a feature of the invention, a skilled molecular biologist or protein chemist may use different break points and different overlapping regions to reduce or increase protease activity, basal luciferase activity or reconstituted luciferase activity to construct a permuted luciferase variant. See Rachel B.Kapust, et al, Biochemical & Biophysical Research Communications, 294(2002) 949-.

Proteases are well known to those skilled in the art and can be selected from a variety of sources, such as bacteria, yeast, fungi, plants, insects, mammals, and the like. The organism requires proteases to process the peptides and, therefore, the biological world provides a wide variety of proteases suitable for use in the present invention. The choice of an appropriate cleavage site for a desired enzyme can generally be found in the literature or in the catalogues of products. Such protease cleavage sites are oligopeptides of various lengths, such as two amino acids, three amino acids, four, five, six, seven, eight, nine, ten or more amino acids, and the like.

The rearranged reporter activating protein may also be substituted with alternative protease cleavage sites or linked to one or more inactive zymogen precursors that can be converted into active enzymes after cleavage. For example, the cleavage site of the proenzyme precursor may be modified to be sensitive to an enzyme that recognizes a sequence different from the wild-type sequence. Alternatively, the cleavage site may also be modified to obtain particularly desirable effects, such as greater specificity, greater sensitivity to cleavage, etc.

The assay can also be performed with an active enzyme having a protease cleavage site, which is converted to an inactive enzyme after cleavage. This feature is somewhat simpler, since multiple proteolytic enzymes with different specificities can act on the active enzyme as desired, with little or no reconstitution.

Mammalian cells, such as HEK293, COS-7, NIH3T3, etc., as well as yeast cells can be used to establish protein-protein interaction rearrangement assays for reporter-activated proteins. Cell-free systems may also be used. Such cell-free systems include lysates, membrane preparations, viral stocks, virus-like particles, liposomes, platelets, membrane preparations, cochleates, other artificially prepared lipid-based units or packages that stimulate biological membranes to form a structure capable of enclosing, attaching, carrying, containing a biological entity such as a transmembrane protein. Organisms such as transgenic organisms may also be used in the present assay, or may be capable of providing cells or reagents useful in the present assay.

The assay of the invention also provides a detectable reporter. The reporter is a substrate for the reporter activating protein under study. Thus, in the case of a permuted luciferase, a suitable reporter molecule is luciferin which, upon luciferase action, produces a detectable luminescent signal. The reporter molecule may also be intracellular, thereby providing an assay that avoids cell lysis. For example, GFP fused to the carboxy terminus of Maltose Binding Protein (MBP) is non-fluorescent when the MBP signal sequence is present. When the MBP signal peptide is removed, fluorescence is observed. See Feilmeier et al, J Bacteriol182(14) 4068-. Thus, as taught herein, a protease cleavage site can be introduced downstream of the MBP signal peptide to create an assay that can be used with living cells.

The assay may utilize rearranged luciferase or fluorescent proteins to monitor subcellular location and displacement of protein-interaction complexes. Figure 4 is a schematic representation of such an embodiment.

The present invention relates to methods for determining whether an agent of interest modulates the interaction between: i) a first protein, such as a membrane bound protein, e.g., a receptor, such as a transmembrane receptor, and ii) a second protein, such as an intracellular molecule, another transmembrane protein, etc., e.g., a member of the arrestin family. One method involves co-transformation or co-transfection of a cell, which may be prokaryotic or eukaryotic and contains two constructs. The first construct comprises a first nucleic acid encoding: (a) a first protein, such as a transmembrane receptor and (b) a cleavage site for a protease, and (c) a second nucleic acid encoding a protein capable of activating the reporter. The second construct comprises (a) a nucleic acid encoding a second protein whose interaction with the first protein is measured and/or determined, and (b) a nucleic acid encoding a protease, a portion of a protease, or a polypeptide having protease activity and acting at the cleavage site of the first construct. In certain embodiments, one or more such constructs may be stably integrated into a cell.

Some features of one embodiment of the invention are shown in figure 1. Briefly, a cell expressing the first protein of interest is obtained. The protein of interest may include a proteolytic moiety, or the proteolytic moiety may bind to the complex upon binding or release of the bound ligand. In response to a change in the ligand binding profile, an inactive enzyme is linked to a peptide moiety which binds to the first protein of interest. In this embodiment, the proximity of the protease to the inactive enzyme allows reconstitution of the enzyme activity, such as luciferase activity. Reconstituted activity has been reported to affect protein-protein interactions.

The example shown in FIG. 1 shows a transmembrane protein, a TEV cleavage enzyme, a permuted luciferase and a luciferase substrate, such as luciferin. In this case, protein "A" may be an inhibitor protein. The first protein of interest may be a GPCR. The N-and C-termini of the luciferase may be rearranged and ligated to a TEV protease cleavage site to produce an inactive rearranged luciferase. The rearranged luciferase shown is fused to beta arrestin 2.

Protein a may be fused to a protease. Protein B can be fused to an inactive rearranged reporter activating protein. The protease recognition and cleavage site (recognized by a protease fused to protein A) is inserted into the rearranged reporter activating protein. Protein a and protein B are brought into proximity with each other by, for example, a third molecule that regulates the interaction of protein a and protein B. The rearranged, inactive reporter activating protein is hydrolyzed by a nearby fusion protease, resulting in the fusion of two fragments of the rearranged, inactive reporter activating protein, thereby regenerating the active reporter activating protein activity. The activity of the reporter activating protein can be assessed using appropriate reagents, devices, using an appropriate reporter such as luciferin using commercially available reagents and kits.

GPCRs as protein a may be fused to TEV protease. Alternatively, the GPCR may also be fused to a rearranged reporter activating protein.

In FIG. 1, one molecule that binds to a GPCR results in the interaction of a β -arrestin with the GPCR. The cleavage site within the rearranged luciferase is hydrolysed by the TEV protease attached to or in the vicinity of protein a to produce a luciferase protein fragment. These fragments reconstitute an active luciferase, the presence of which may be detected or inferred using a suitable reporter molecule capable of measuring luciferase activity, such as luciferin in the cell or lysate.

The method can produce a specific signal for a receptor protein such as a GPCR interacting with a G protein or a β arrestin.

The general approach shown in figure 1 is generally applicable to GPCRs, as β arrestin recruitment is a common phenomenon. However, any pair of molecules that interact, bind or associate, or the like, or are suspected of interacting, binding, associating, or the like, can be used in the practice of the present invention.

One exemplary method employs the β arrestin signaling pathway and does not require prior knowledge of specific G-protein couplings, as this assay is not specific to the GPCR or to the G-protein involved. Thus, this assay is ideal for orphan GPCRs, where the G protein coupling pathway is unknown. This method produces an immediate and physiologically relevant reading without the need for transcriptional amplification as in U.S. patent 7,049,076 (Lee et al).

These materials and methods also enable monitoring of G protein independent phenomena. In this case, the rearranged reporter activating protein can be used to label the β -arrestin. A molecule suspected of or known to interact with a betaarrestin may be labelled with an appropriate protease, such as a GPCR that shows a preference for betaarrestin.

Complementation assay with respect to enzyme fragment (e.g., DiscoverX PathHunter)^TMBetaarrestin assay) the present invention is advantageous because in an enzyme fragment complementation assay, the interacting partners must be joined or brought together to ensure complementation of the enzyme fragments. On the other hand, in the methods of the invention, once proteolysis occurs due to reagent proximity, an active reporter activator is generated and meaningful analysis is achieved without the need for the protein interaction partner to remain bound.

The nucleic acid encoding this first fusion protein and other peptide components can be introduced into a host cell. Such cell modifications are well known in the art. The nucleic acids of the various peptides can be constructed as single molecules, or introduced sequentially or simultaneously. For example, certain constructs may be integrated into the host chromosome to obtain stable transfection, and the materials and methods used are known in the art.

In another alternative system, the two proteins of interest may interact in the absence of a ligand or test compound. The ligand and test compound may cause the two proteins to dissociate, change conformation, or reduce or inhibit their interaction. In this case, the level of free, functionally active proteolytic enzyme in the cell is reduced in the presence of a positive test compound, resulting in a reduced degree of hydrolysis and a significant reduction in the activity of the reporter activating protein.

In a typical embodiment, the arrestin is a second protein that binds to a transmembrane receptor in the presence of an agonist; however, it will be appreciated that since the receptor is only one type of protein, the assay is not dependent on the use of receptor molecules, nor is agonist binding the only interaction that can occur. Any protein that interacts with a second protein may be used, although transmembrane proteins are of interest because they elicit cellular, organ, and tissue responses when receptors are exposed to a modulator that accelerates the cell-bound receptor into an active state. Furthermore, binding of an agonist to a receptor is not the only type of binding that can be assayed. Inverse agonists can also be assayed using the assay. The antagonists themselves may be assayed according to the methods of the invention, as may the relative intensities of the different antagonists and/or agonists.

Additional details of the invention, including specific methods and techniques of making and using the subject matter described herein, are set forth in the description below.

Products that are features of the invention may also be described simply as methods described herein. For example, in a "three-part construct", i.e., a construct containing sequences encoding: i) a protein, ii) a cleavage site, and iii) a reporter activating protein; the protein may be, for example, an intracellular protein or a membrane bound protein, such as a transmembrane receptor, for example, a member of the GPCR family. The cleavage site may be any hydrolysable site, the hydrolysis of which may be effected by the action of a protease of a partner protein of a protein-protein interaction. Cleavage can directly generate the reporter, or cleavage can also cause rearrangement of the reporter-activating protein, thereby generating the reporter from another molecule. The third portion may be a protease or a polypeptide having protease activity.

These sequences can be modified so that the C-terminus of the protein they encode interacts better and stronger with a second protein. For example, such modifications may include the replacement of the C-terminal coding sequence of the protein, such as the GPCR, with the C-terminal coding region of AVPR2, AGTRLI, F2PLI, CCR4, CXCR2/IL-8, and the like. The gene sequence may be recoded to optimize translation of the protein of interest in the host cell.

The protein that activates the reporter may be one that acts within the cytoplasm or an organelle such as the nucleus, or it may be one that initiates a cascade of reactions that cause another protein to act. Such cascade reactions are well understood by those skilled in the art as they are cellular events that have been extensively studied. For example, a translocation signal, such as a nuclear translocation sequence, can be incorporated into the reporter enzyme. Localization sequences are well known in the art.

The second construct, as described above, includes a region encoding a protein that interacts with the first protein, resulting in some measurable phenomenon. The protein may be an activator, competitor, inhibitor, or a component that produces a synergistic response, or more broadly, a "modulator" of the first protein. Members of the arrestin family are exemplary, particularly when the first protein is a GPCR, however, other protein coding sequences may be used, particularly when the first protein is not a GPCR. The second part of these two-part constructs encodes a protease, a portion of a protease, or a polypeptide having protease activity that cleaves the reporter activating protein encoded by the first construct to generate a reporter activating protein that directly or indirectly generates a detectable signal.

However, these exemplary embodiments do not limit the invention, as discussed in other embodiments below, e.g., a protease may be fused to protein A or protein B as a design choice.

Host cell

The terms "cell", "cell line", "cell culture" and "cell culture" are used interchangeably herein. A host cell may also refer to a source cell from which a lysate may be obtained. All of these terms also include their progeny, i.e., any and all progeny. It is understood that all progeny are not identical due to deliberate or inadvertent mutation, selection, or differentiation. The host cell can be engineered to express a screenable or selectable marker or reporter that emits a signal when acted upon by the reporter activating protein of the first construct cleaved by a protease that is part of the fusion protein of the second construct. The screenable marker or reporter may be introduced into the host cell or assay system in any manner.

Numerous cell lines and cell cultures can be used as host cells. For example, many host cells are available from the American Type Culture Collection (ATCC), a facility for the preservation of living cultures and genetic material. The appropriate host cell can be determined by one skilled in the art based on the vector backbone characteristics and the desired results. For example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate many vectors. Cell types that can be used for vector replication and/or expression include, but are not limited to, bacteria such as E.coli (e.g., E.coli strain RR1, E.coli LE392, E.coli B, E.coli X1776(ATCC No.31537), E.coli W3110 (F)^-，lambda^-Prototroph, ATCC No.273325), DH5 α, JM109 and KC8), bacillus such as bacillus subtilis; and other enteric bacteria such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas species, and some commercially available bacterial hosts such asCompetent Cells and SOLOPACK^TM Gold Cells(LaJolla). In certain embodiments, bacterial cells such as E.coli LE392 can be used as host cells for phage viruses.

Examples of eukaryotic host cells for vector replication and/or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293(HEK), COS, CHO, Saos, and PC 12. Other cells, such as yeast cells and insect cells, such as Sf9 cells, are also suitable. Depending on the purpose of use, a person skilled in the art can select the host cell he or she wishes to use. Those skilled in the art will recognize that there are many host cells available from a variety of cell types and organisms.

Similarly, viral vectors (including bacteriophages) can be used in conjunction with eukaryotic or prokaryotic host cells, particularly cells that allow for replication or expression of the vector. The host cell need not be an immortalized cell line. The host cell may be derived from a stem cell culture or a primary cell culture, such as hematopoietic stem cells, blood vessels, epithelium, smooth muscle, spleen, T cells, B cells, monocytes, and the like. The host cell may be transgenic, such as containing genetic material of another organism. Cells that cannot be used in the Lee et al method can also be used in the assay methods of the invention, since the methods of the invention do not require active transcription. For example, enucleated cells such as red blood cells or platelets can be used in the present invention.

In the assays of the invention, host cells are intended to include artificial packages and units, such as liposomes and virus-like particles. Such structures typically mimic or mimic a cell or portion thereof, creating an enclosed structure with an internal cavity completely or partially isolated from the exterior by a membrane or other structure. As noted above, such artificially prepared packages and units include liposomes, cochlear bacteria, virus-like particles, viruses, and the like.

Protein

The present invention contemplates the use of any two proteins known or suspected to have a physical interaction. In certain embodiments, the proteins are in the form of fusion proteins or are configured in the form of fusion proteins, a first protein fused to a latent or inactive reporter activating polypeptide, and a second protein fused to a protease that recognizes a cleavage site on the first fusion protein, wherein cleavage of the first fusion protein releases or renders active the reporter activating polypeptide.

As regards the first protein studied, it may be a naturally occurring membrane-associated protein, or it may be constructed so as to be membrane-associated. For example, the first protein may be a transmembrane receptor, such as a GPCR, or any other transmembrane receptor of interest, including but not limited to receptor tyrosine kinases, receptor serine/threonine kinases, cytokine receptors, and the like. Furthermore, it is well known that certain portions of a protein will function in the same manner as a full-length first protein, and that active portions of such first protein, such as the extracellular or transmembrane domains, are within the definition of protein herein.

It will be apparent to those skilled in the art that the present invention can be used to assay for interaction with any protein, and is not limited to assaying only membrane bound receptors, such as GPCRs. For example, the activity of other classes of transmembrane receptors, including but not limited to: receptor Tyrosine Kinases (RTKs), such as IGF1R, such as Epidermal Growth Factor Receptor (EGFR), ErbB2/HER2/Neu or related RTKs; receptor serine/threonine kinases, such as transforming growth factor-beta (TGF β), activin or Bone Morphogenic Protein (BMP) receptors; cytokine receptors, such as receptors of the interferon family of interleukins, erythropoietin, G-CSF, GM-CSF or Tumor Necrosis Factor (TNF); a leptin receptor; and other receptors that do not generally need to be membrane bound, such as estrogen receptor 1(ESR1) and estrogen receptor 2(ESR 2). In each case, the method may involve transfecting the cell with a modified receptor polynucleotide that directs the expression of a chimeric or fused protein, including the receptor of interest, a protease cleavage site, and a reporter activating polypeptide. The cell may be co-transfected with a second polynucleotide, such as a vector, which directs the expression of a chimeric or fused protein, including an interacting protein fused to a protease that recognizes and cleaves the first protein cleavage site. The first and second polynucleotides may be included within a single molecule, thereby avoiding co-transfection. In the case of a RTK, such as EGFR, this interacting protein may consist of SH2(Src homology domain 2) containing a polypeptide such as phospholipase C (PLC) or Src homology 2 domain containing the transforming protein 1(SHC 1). In the case of receptor serine/threonine kinases, such as TGF β, activin and BMP receptors, this interacting polypeptide may be a Smad protein or a portion thereof. In the case of cytokine receptors, such as the interferon alpha, interferon beta or interferon gamma receptor, this interacting protein may be a signal transduction and transcriptional activation (STAT) protein, such as but not limited to STAT1 or STAT 2; or a Janus kinase (JAK) protein, JAK1, JAK2, or Tyk 2; or portions thereof, and the like. The transfected cell may contain a reporter molecule that is acted upon by a reporter activating protein. An assay is then performed in which the transfected cells are treated with a test compound for a certain period of time and, at the end of the test, the activity of the reporter is measured. If the test compound activates the reporter of interest, it will trigger an interaction between the receptor of interest and the interacting protein, resulting in cleavage of the protease cleavage site and activation of the reporter activating protein, resulting in a measurable change or increase in reporter activity.

Other possible protein pairs include antibody-antigen, enzyme-substrate, dimerizing protein, signal transduction cascade components, components of complex structures such as ribosomes or viruses, intercellular interactive molecules on different cells such as antigen presenting cells and immune cells for response such as T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, and the like, as well as other protein pairs known in the art. As to which protein, e.g., A or B, binds to or dissociates from, proteases and proteins with protease recognition sites are interchangeable.

Reporter molecules

The reporter may be any molecule that changes appearance or function in response to the activity of the active reporter activating molecule and produces a detectable signal or can be easily monitored to track such changes. These terms are intended to be applied loosely. Reporter activating proteins, once activated (or in some possible embodiments inactivated), will produce a detectable change in the reporter. Detection of such a change can be used to determine, for example, whether a test compound has modulated protein-protein interactions. Other non-enzymatic reporter activating proteins may be used so long as they produce a detectable signal. Thus, certain known reporter activating proteins, such as galactosidase, peroxidase, luciferase, and the like, can be used. Certain known reporter molecules, such as galactosidase substrates, peroxidase substrates, luciferase substrates, GFP, and the like, can also be used.

Proteases and cleavage sites

Proteases are well-characterized enzymes that cleave other proteins at specific sites. One protease family, the Ser/Thr protease family, can cleave at serine and/or threonine residues. Other proteases include cysteine or thiol proteases, aspartic proteases, metallo proteases, aminopeptidases, di-and tri-peptidases, carboxypeptidases and peptidyl peptidases. The choice of these enzymes is determined by one skilled in the art and is not necessarily limited to the molecules described herein. It is well known that enzymes have catalytic domains that can be used to replace full-length proteases. These matters are also included in the present invention. One particular embodiment is tobacco etch virus nuclear inclusion a protease (TEV), or an active portion thereof. As will be appreciated by those skilled in the art, other specific cleavage sites for proteases may be used.

Modification of proteins

In some embodiments of the assay, the first protein may be modified to enhance its binding to the affected protein. For example, certain GPCRs are known to bind to profilins more stably or with greater affinity when stimulated by ligands, and this enhanced interaction is mediated by isolated domains, such as clusters of C-terminal tail serine and threonine residues (Oakley, et al, J.biol. chem., 274: 32248-32257, 1999and Oakley, et al, J.biol. chem., 276: 19452-19460, 2001). Using this example, it is clearly understood that the receptor coding sequence itself may be modified to increase the affinity between a membrane-bound protein, such as a receptor, and the protein to which it binds. An example of these changes is the modification of the C-terminal region of membrane-bound proteins such as 7TMR, which may involve replacing a portion of it with a region corresponding to another receptor that has higher affinity for the binding protein but does not affect the receptor binding function.

Additionally or alternatively, the second protein may also be modified to enhance its interaction with the first protein. For example, the assay may incorporate point mutations, truncations or other variants of a second protein, such as an arrestin, which is known to bind to agonist-occupied GPCRs more stably or in a manner independent of phosphorylation (Kovor, et al, J.biol.chem., 274: 6831-6834, 1999). Such modifications can be carried out using methods known in the art.

Assay format

In several embodiments, the present invention provides a simple way to assess the interaction between two proteins expressed in the same cell, unit or reaction mixture. The first construct may comprise a sequence encoding a first polypeptide linked to a polynucleotide encoding a protease, protease moiety or cleavage site for a polypeptide having protease activity, which is itself linked to a polynucleotide encoding a reporter enzyme. The term "linked to" describes a situation in which the sequences are fused to form a single, complete open reading frame that can be translated into a single polypeptide containing all the elements. These may, but need not, be separated by additional nucleotides, which may or may not encode additional proteins or peptides. The second construct inserted into the recombinant cell may comprise both a polynucleotide encoding a second protein and a protease, protease moiety or polypeptide encoding protease activity. These essential elements combine to form a basic assay format when combined with a candidate agent whose effect on target protein interaction is to be determined.

However, by using different reporter molecules simultaneously, the invention can also be used to assay more than one membrane bound protein such as receptors, each activated by activation of a different protein species as described herein. This can be achieved, for example, by mixing cells transfected with different receptor constructs and different reporter activating proteins, or by fusing different enzymes to each test receptor and measuring the activity of each reporter gene upon treatment with the test compound. For example, it may be desirable to determine whether the molecule under investigation activates a first receptor and whether side effects should be expected as a result of interaction with a second receptor. In this case, it is possible to refer to, for example, a first cell line encoding a first receptor and a first receptor activating protein, such as lacZ, and a second cell line encoding a second receptor activating protein and a second receptor, such as GFP. In that case, the GFP can be rearranged as practiced in the present invention. The two cell lines can be mixed and the compound under study added and then positive results can be sought for one cell line without affecting the other.

Alternative forms of the invention relate both to assays which test only a pair of interacting proteins and to what is referred to herein as a "multiplex" assay. Such assays can be performed in a variety of ways, but in all cases more than one pair of proteins are assayed simultaneously. This can be achieved by providing more than one sample of cells, each sample being transformed or transfected to analyse each pair of interacting proteins. Different transformed cells can be pooled in the same vessel and tested simultaneously, or each different transformant can be placed in a different well and tested. Alternatively, the cells may be treated to carry a first protein with multiple labels, such as transmembrane-based proteins, to determine whether a ligand or candidate molecule can activate more than one receptor.

The cells used in the multiplex assays described herein may be, but need not be, identical. Similarly, the reporter system used in each sample may be, but need not be, the same. After a sample is placed in a container, such as a well of a microarray, one or more compounds can be screened against multiple pairs of interacting proteins in the container.

FIG. 10 shows the general results obtained with the present analytical method. At low or high concentrations (depending on whether the modulating effect is inhibitory or activating), the test compound may not be effective. As the concentration of the test compound decreases or increases, the modulation may change. A dose response curve as shown in figure 10 can be used to assess the modulating effect. A single point may also be evaluated. For example, the single point may be a predetermined value different from the control or background. The predetermined value is typically determined on a statistical basis by accumulating certain data or making multiple measurements of samples from "normal" subjects to obtain a population average of samples with standard errors and standard deviations. A constant may be used as a predetermined different value. Generally, ratios will be used, such as at least 10% off control samples, but more often times multiples of control samples, such as about 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more (or the inverse thereof) times the control value predetermined in another analysis. The predetermined threshold indicative of adjustment is typically calculated by one skilled in the art based on the situational cues or needs, balancing the type 1 and type 2 errors as dictated or required by the situation.

Reagent kit

Any of the compositions described herein and any combination thereof can be provided in the form of a kit. Thus, a kit will comprise one or more components, such as a vector or cell of the invention, and any other reagents that may be used in accordance with the invention, in a suitable container.

The kit may comprise one or more compositions of the invention in suitable aliquots. The components of the kit may be provided in aqueous medium or in lyophilized form or as a concentrate of the solute in a suitable solvent. The containers of the kit generally comprise at least one vial, test tube, volumetric flask, bottle, syringe or other container, which may be filled or already filled with one of the components, and preferably already suitably aliquoted. Where the kit includes more than one component, the kit will typically further include a second, third or other additional container for separately containing the other components. However, various combinations of components may be contained in a single container, such as a vial. In addition, a suitable diluent may also be provided. The kits of the invention will also typically include means for packaging the reagent containers for commercial sale. Such containers may include injection molded or blow molded plastic or foam containers to hold the required reagent bottles and instructions.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution can be an aqueous solution, such as a particularly useful sterile aqueous solution. However, the components of the kit may also be provided in the form of a dry powder or attached to a solid support. When the reagents and/or components are provided in the form of a dry powder, reconstitution can be carried out by adding a suitable solvent to the dry powder. It is envisaged that the solvent, such as sterile water or a suitable saline or buffer solution, may also be contained in another container.

Examples

Specific examples illustrating embodiments of the present invention are given below, but the present invention should not be construed as being limited to these examples only.

Example 1

Figure 1 shows an embodiment comprising a permuted inactive luciferase whose activity is reconstituted by the action of TEV protease on the TEV protease recognition site comprised. As shown, the first protein is fused to a protease. Example 1 is designed to use the activity of TEV protease to reconstitute the activity of a permuted luciferase as an embodiment of the second protein. As shown, the second protein is fused to an inactive rearranged reporter activating protein, luciferase. A protease recognition and cleavage site recognized by a protease fused to the protein of interest is inserted into the rearranged reporter activating protein. The first and second proteins are brought into proximity with each other by a third molecule that modulates the interaction between the first and second proteins. Hydrolysis of the rearranged, inactive reporter activating protein by a nearby fusion protease results in cleavage of the rearranged, inactive reporter activating protein to form two fragments, thereby regenerating the active reporter activating protein. The activity of the reporter activating protein can be assessed using appropriate reagents and devices.

The permuted luciferase was constructed by rearranging firefly luciferase N-terminal amino acids 2 to 233 and C-terminal amino acids 234 to 550 in reverse order, interrupted by a TEV protease recognition site ENLYFQX (SEQ ID NO: 3). Cleavage at this site results in the reconstituted activity of the rearranged luciferase. The position of X can be any amino acid that determines the recognition affinity and cleavage efficiency of TEV protease. Alteration of X has been shown to modulate the enzymatic kinetics of TEV. Similar amino acid substitutions at other positions in the recognition sequence can also alter enzyme kinetics. Modulation of kinetics is advantageous for optimizing, for example, incubation time during screening and background activity affecting signal/noise parameters. The permuted luciferase (luc234X233, where X is the specific amino acid N-terminal to the TEV heptapeptide cleavage site, SEQ ID NO: 3) was then fused to the C-terminus of one GPCR, ADRB2, to generate a GPCR-permuted luciferase ADRB2-luc234X233, an expression plasmid.

Example 2

Human beta arrestin 2-TEV fusion plasmids were constructed by fusing tobacco etch virus protease A to the C-terminus of beta arrestin 2. All DNA fragments were generated by PCR using the appropriate template. The GPCR-luc234X233 fusion gene was subcloned in pcDNA3.1(+) (Invitrogen) with a neomycin selection marker, and the Arr-TEV fusion gene was subcloned in pcDNA3.1(+) (Invitrogen Cat. #43-0018) with a bleomycin (zeocin) selection marker.

Example 3

CHO-K1 cells were co-transfected with ADRB2-luc234R233 (example 1) and Arr-TEV plasmid (example 2) using an appropriate commercially available transfection kit. 48 hours after transfection, cells were treated with or without 10. mu.M ADRB2 agonist isoproterenol for 2 hours, and Bright-GLO was added to the cells^TMOr Steady-GLO^TM(Promega) and the relative luminescence of the lysates was recorded with a suitable luminescence reader. In the presence of isoproterenol, a more than three-fold increase in luminescence activity was observed.

FIG. 5 shows GPCR/permuted luciferase expression with or without addition of Arr 2-TEVp.In the data shown in the figure, constructs were introduced into cells, however, transfected cells were not contacted Any modifier. Thus, the data indicate that there is some spontaneous cleavage when the cleavage site contains serine Active, but when X is R or V, substantially no background noise appears. As indicated in fig. 6 As shown, a response is observed when cells expressing R or V cleavage sites are exposed to an agonist.FIG. 7 shows dose-dependent response of luciferase activity in cells transiently and stably expressing GPCR-luc234V233 and/or Arr-TEV.

Example 4

FIG. 8 shows that incubation times of 5 hours or 1 hour were sufficient to analyze protein-protein interactions. The dose response relationship is clearly shown in the figure.

An ADRB2-TEV fusion gene expression plasmid was constructed by fusing tobacco etch virus protease A to the C-terminus of ADRB2 and inserting the fused gene into pcDNA3.1(+) with zeocin selection marker (Invitrogen Cat # 43-0018). All DNA fragments were generated by PCR using appropriate templates known in the art.

A β arrestin 2-rearranged luciferase (Arr-luc234X233) fusion gene expression plasmid was constructed by fusing the rearranged luciferase luc234X233 to the C-terminus of β arrestin 2. The TEV protease cleavage site is ENLYFQ/X, (Rachel B.Kapust, et al Biochemical & Biophysical Research Communications, 294(2002)949-955), where E and Q are generally invariant, where X can be any amino acid, although G and S are amino acids that are common at the cleavage site. Cleavage occurs between the Q and X residues. X may determine the efficiency of the cleavage. In certain embodiments, the TEV luciferase cleavage site is included in a permuted luciferase. Background and signal/noise ratio can be improved using simple routine experimentation. For example, it has been found that in certain applications, replacing glycine with valine at the X hydrolysis site of TEV can reduce background. The fused fusion gene was cloned in pcDNA3.1(+) using a neomycin selection marker (Invitrogen).

HEK293 cells were co-transfected with ADRB2-TEV, the TEV recognition sequence of ENLYFQV (SEQ ID NO: 12), and Arr-luc234V233 plasmids using an appropriate commercially available transfection kit. 48 hours after transfection, cells were treated with varying concentrations of ADRB2 agonist isoproterenol for 1 hour and 5 hours, and Bright-GLO was added to the cells^TM(Promega) and in Envison II^TMRelative luminescence units are recorded above. After 1 and 5 hours incubation with isoproterenol, a dose-dependent luminescent activity was observed.

Example 5

FIG. 9 shows, on the left, ligand-induced luciferase activity in HEK293 cells stably expressing Arr-luc234V233 and transiently expressing ADRB2-TEV fusion protein. The right panel shows stable expression of the arrestin reporter activating protein construct and transient expression of the 7 TMR-protease fusion in CHO cells.

In HEK293 or CHO cells, stable cell lines expressing GPCR-luc234R233 or Arr-TEV were generated. HEK293 and TranIT-CHO cells were transfected with Lipofectamine at 20 ng/well per DNA in a 12-well plate.

In per-luc assays, 384 well plates are typically used. Other plate forms are also acceptable. CHO cells stably expressing GPCR-luc234R233 or Arr-TEV were seeded at 10,000 cells per well on a surface 384-well white assay plate (Becton Dickinson) treated with tissue culture. The next day, cells were treated with agonist at a concentration of 10. mu.M to 0.7pM (serial 3: 1 dilution in serum-free cell culture medium). The Luciferase activity was measured using the Steady-Glo Luciferase assay System (Promega). After 2 hours of agonist treatment, the culture was aspirated and 25. mu.l luciferase assay reagent was added to each well. Relative Luminescence Units (RLU) were read using a multi-tag reader EnVision from Perkin Elmer. Data were plotted and analyzed using PRlSM software.

HEK293 cells stably expressing Arr-luc234V233 were generated by selection for neomycin tolerance. The neomycin-resistant gene is present in the Arr-luc234V233 expression plasmid vector pcDNA3.1.

Example 6

FIG. 9 shows the response of isoproterenol to dose in CHO cell lines to the right.

In CHO cells, stable cell lines expressing GPCR-luc234R233 or Arr-TEV were generated. Transfection was performed using the TransfectIT-CHO transfection kit (Mirus Bio, Madison, Wis.) in a 12-well plate at 1. mu.g of each DNA per well. Individual colonies were harvested from the transfectants as selective for neomycin and zeomycin.

Arr-luc234V233 stably expressing cells were transfected with ADRB2-TEV plasmid using an appropriate commercially available transfection kit. Cells transiently expressing ADRB2-TEV and stably expressing Arr-luc-234V233 were cultured with isoproterenol for 2 hours, and Bright-GLO was added to the cells^TMA luciferase reagent. Dose dependent luciferase activity was recorded on EnVison II.

Example 7

FIG. 10 shows the evaluation of agonist, partial agonist, antagonist and nonspecific endogenous receptor responses using GPCR Per-Luc assays.

HEK293 cells stably expressing Arr-luc234V233 were transfected with ADRB2-TEV plasmid using Lopofectamine2000 transfection reagent (Invitrogen). 48 hours after transfection, different concentrations of the known agonist isoproterenol; cells were cultured for 2 hours with the partial agonist BRL37344(Sigma-Aldrich), the antagonist ICI118551(ICI), the antagonist ICI118551 with 200nM isoproterenol, and the agonist S1P (sphingosine-1-phosphate) of the HEK293 endogenous EDG receptor, and then Bright-GLO was added to the cells^TMA luciferase reagent. Dose dependent luciferase activity was recorded on EnVison II as shown in figure 10.

EC50 and IC50 values for this assay were similar to those obtained from FLIPR and cAMP assays. The endogenous receptor EDG and its ligand S1P in HEK293 cells did not affect luciferase activity, whereas other assays, such as FLIPR and cAMP, produced positive signals. The agonist isoproterenol produces a response. The partial agonist BRL37344 produces a gradual response. The antagonist ICI18551 inhibited isoproterenol, but was inactive alone. Thus, the assay method of the invention is specific, as shown in FIG. 10 (considered in combination with other comparable data), yielding less and lower false positive signals.

Example 8

FIG. 14 shows an example in which a permuted luciferase was constructed by cloning N-terminal amino acids 2 to 456 of firefly luciferase behind C-terminal amino acids 234 to 550 using a TEV protease recognition site ENLYFQX with V as X. This permuted luciferase (luc234V456) was fused to the C-terminus of the GPCR, ADRB2, to produce a GPCR-permuted luciferase construct, ADRB2-luc234V456 expression plasmid.

All DNA fragments were generated by PCR using the appropriate template. The ADRB2-luc234V456 fusion gene was cloned in pcDNA3.1(+) using a neomycin selection marker (Invitrogen).

CHO-K1 cells were co-transfected with ADRB2-luc234V456 and Arr-TEV plasmid using an appropriate commercially available transfection kit. 48 hours after transfection, cells were treated with and without 10. mu.M ADRB2 agonist isoproterenol for 2 hours, to which Bright-GLO was added^TMOr Steady-GLO^TM(Promega) and relative luminescence was recorded with a suitable luminescence reader. Reconstituted luciferase activity was observed for different doses of isoproterenol.

HEK293 cells stably expressing Arr-luc234V233 were selected for tolerance to neomycin. The neomycin-resistant gene is present in the Arr-luc234V233 expression plasmid vector pcDNA3. Luciferase activity in response to agonist was observed.

Example 9

Figure 13 shows the dose dependence of V2 inverse agonist.

In this example, HEK293 cells stably expressing Arr-luc234V233 were transfected with V2-TEV plasmid using Lipofectamine2000 transfection reagent (Invitrogen). 48 hours after transfection, cells were cultured for 2 hours using different concentrations of SR121463(sanofi recheche, Toulose, FR) which is a compound considered an antagonist by standard methods. Bright-GLO^TMLuciferase was added to the cells. Dose dependent luciferase activity was recorded on EnVison II. As the amount of SR121463 (more appropriately defined as an inverse agonist) increases, the level of luminescence also increases.

In this assay, inverse agonists behave like other inverse agonists. Inverse agonists are known to block the signaling pathway of the V2G protein and to promote beta-arrestin-mediated activation of MAPK (Azzi et al, PNAS, 2003, 100: 11406-11411). Thus, this assay of the invention can indicate a unique active conformation of the G protein-coupled receptor.

In contrast, in classical assay systems, inverse agonists of GPCRs exhibit antagonist behavior. This is because inverse agonists may bind to and stabilize the inactive conformation of GPCR G protein signaling. However, certain inverse agonists both stabilize inactive forms of GPCRs for G protein signaling and also promote recruitment of β -arrestins to GPCRs to activate β -arrestin signaling pathways.

Example 10

FIG. 6 shows agonist-induced luciferase activity.

In this example, a permuted luciferase was constructed by rearranging firefly luciferase N terminal amino acids 2 to 233 and C terminal amino acids 234 to 550 in reverse order, separated by a TEV protease recognition site, ENLYFQX. Position X may be any amino acid. The amino acid at this position is known to determine the recognition affinity and cleavage efficiency of TEV protease. This rearranged luciferase (luc234X233) was then fused to the C-terminus of the GPCR, ADRB2, to generate a GPCR-rearranged luciferase, ADRB 2-lucucc 234X233 expression plasmid.

Human beta arrestin 2-TEV fusion plasmids were constructed by fusing tobacco etch virus protease A to the C-terminus of beta arrestin 2. DNA fragments were generated by PCR using the appropriate template. The GPCR-luc234X233 fusion gene was subcloned in pcDNA3.1(+) using the neomycin selection marker (Invitrogen) and the Arr-TEV fusion gene was subcloned in pcDNA3.1(+) (Invitrogen Cat. #43-0018) using the zeocin selection marker.

CHO-K1 cells were co-transfected with ADRB2-luc234R233 and Arr-TEV plasmid using an appropriate commercially available transfection kit. 48 hours after transfection, cells were treated with or without 10. mu.M ADRB2 agonist isoproterenol for 2 hours, and Bright-GLO was added to the cells^TMOr Steady-GLO^TM(Promega) and use the appropriate hairThe relative luminescence was recorded by the optical reader. In the presence of isoproterenol, a more than three-fold increase in luminescence activity was observed.

Example 11

Figure 12 shows the results of agonists, antagonists, and inverse agonists using the invention (left) and another assay (right). The two figures show the difference and inverse effect of agonist, antagonist and inverse agonist. The methods of the invention provide good specific activity.

Example 12

FIG. 7 shows CHO cells harboring ADRB 2-rearranged luciferase and Arr-TEV. The ADRB2-luc234V233 data in the left panel were processed to contain the TEV recognition site ENLYFQV. CHO-K1 cells were co-transfected with ADRB2-luc234V233 and Arr-TEV plasmids using an appropriate commercially available transfection kit. 48 hours after transfection, cells were treated with varying concentrations of ADRB2 agonist isoproterenol for 2 hours, and Bright-GLO was added to the cells^TMOr Steady-GLO^TM(Promega) and relative luminescence was recorded with a suitable luminescence reader.

Example 13

The right panel of FIG. 7 summarizes data using stably transfected cells with different cleavage sites. The results are similar to the left. Thus, both GPCR-luciferase constructs with different cleavage sites respond to agonists.

Example 14

FIG. 6 shows agonist-induced signaling activity comparing X for R and X for V. The results are similar, indicating that X can vary in general.

In this example, a permuted luciferase was constructed by rearranging firefly luciferase N terminal amino acids 2 to 233 and C terminal amino acids 233 to 550 in reverse order, separated by a TEV protease recognition site, ENLYFQ/X. Located at X may be any amino acid that determines the recognition affinity and cleavage efficiency of the TEV protease. V and R are shown. The permuted luciferase (luc234X233) was fused to the C-terminus of the GPCR, ADRB2, to produce a GPCR-permuted luciferase, ADRB 2-lucucc 234X233 expression plasmid.

In this example, a human β arrestin 2-TEV fusion plasmid was constructed by fusing tobacco etch virus protease a to the C-terminus of β arrestin 2. All DNA fragments were generated by PCR using the appropriate template. The GPCR-luc234X233 fusion gene was subcloned in pcDNA3.1(+) (Invitrogen) using the neomycin selection marker, and the Arr-TEV fusion gene was subcloned in pcDNA3.1(+) using the zeocin selection marker (Invitrogen Cat. # 43-0018).

CHO-K1 cells were co-transfected with ADRB2-luc234R233 and Arr-TEV plasmid using an appropriate commercially available transfection kit. After 48 hours, the cells were treated with or without 10 μ MADRB2 agonist for 2 hours, and Bright-GLO was added to the cells^TMOr Steady-GLO^TM(Promega) and relative luminescence was recorded with a suitable luminescence reader. In the presence of isoproterenol, a more than three-fold increase in luminescence activity was observed.

Example 15

FIG. 11 shows, on the left, the results of 8-AVP agonists in cells transiently expressing V2-TEV.

In this example, HEK293 cells stably expressing Arr-luc234V233 were transfected with V2-TEV plasmid using Lipofectamine2000 transfection reagent (Invitrogen). After 48 hours, different concentrations of agonist 8-AVP (Arg)⁸Vasopressin, a known agonist of the V2 vasopressin receptor), and adding Bright-GLO to the cells for 2 hours^TMA luciferase reagent. Dose dependent luciferase activity was recorded on EnVison II.

Example 16

HEK293 cells stably expressing Arr-luc234V233 were transfected with V2-TEV plasmid using Lipofectamine2000 transfection reagent (Invitrogen). After 48 hours, the cells were incubated with different concentrations of inverse agonist for 2 hours and Bright-GLO was added to the cells^TMA luciferase reagent. Dose dependent luciferase activity was recorded on EnVison II.

In this assay, inverse agonists exhibit agonist behavior. It is now known that certain inverse agonists block the signaling pathway of the V2G-protein, promoting beta arrestin-mediated activation of MAPK (Azzi et al, PNAS, 2003, 100: 11406-11411). Thus, the assay can assess the unique active conformation of G protein-coupled receptors.

Example 17

Figure 11, right panel, shows dose-dependent luciferase activity generated by V2 inverse agonist by promoting β arrestin interaction with the V2 receptor.

In this example, HEK293 cells stably expressing Arr-luc234V233 were transfected with V2-TEV plasmid using Lipofectamine2000 transfection reagent (Invitrogen). After 48 hours, the cells were incubated with different concentrations of inverse agonist for 2 hours and Bright-GLO was added to the cells^TMA luciferase reagent. Dose dependent luciferase activity was recorded on EnVison II.

Other features of the present invention will be apparent to those skilled in the art and need not be described in detail herein. Various modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

All documents cited herein are incorporated by reference herein in their entirety.

Sequence listing

<110> H.Esinderleio

Chua Jidong

P, S, white

P-wessense

<120> identification of molecules that modulate protein-protein interactions

<130>US2006/244

<160>15

<170> PatentIn version 3.3

<210>1

<211>27

<212>DNA

<213> tobacco etch Virus

<400>1

ggatccgcag agttgatcat catagtc 27

<210>2

<211>31

<212>DNA

<213> tobacco etch Virus

<400>2

gggcccctat tgcgagtaca ccaattcatt c 31

<210>3

<211>7

<212>PRT

<213> tobacco etch Virus

<220>

<221>SITE

<222>(7)..(7)

<223> peptides recognized by TEV protease were altered. In FIG. 5X is S, R or V.

<400>3

Glu Asn Leu Tyr Phe Gln Xaa

1 5

<210>4

<211>1237

<212>DNA

<213> human adenovirus type 1

<400>4

gggcaacccg ggaacggcag cgccttcttg ctggcaccca atagaagcca tgcgccggac 60

cacgacgtca cgcagcaaag ggacgaggtg tgggtggtgg gcatgggcat cgtcatgtct 120

ctcatcgtcc tggccatcgt gtttggcaat gtgctggtca tcacagccat tgccaagttc 180

gagcgtctgc agacggtcac caactacttc atcacttcac tggcctgtgc tgatctggtc 240

atgggcctgg cagtggtgcc ctttggggcc gcccatattc ttatgaaaat gtggactttt 300

ggcaacttct ggtgcgagtt ttggacttcc attgatgtgc tgtgcgtcac ggccagcatt 360

gagaccctgt gcgtgatcgc agtggatcgc tactttgcca ttacttcacc tttcaagtac 420

cagagcctgc tgaccaagaa taaggcccgg gtgatcattc tgatggtgtg gattgtgtca 480

ggccttacct ccttcttgcc cattcagatg cactggtacc gggccaccca ccaggaagcc 540

atcaactgct atgccaatga gacctgctgt gacttcttca cgaaccaagc ctatgccatt 600

gcctcttcca tcgtgtcctt ctacgttccc ctggtgatca tggtcttcgt ctactccagg 660

gtctttcagg aggccaaaag gcagctccag aagattgaca aatctgaggg ccgcttccat 720

gtccagaacc ttagccaggt ggagcaggat gggcggacgg ggcatggact ccgcagatct 780

tccaagttct gcttgaagga gcacaaagcc ctcaagacgt taggcatcat catgggcact 840

ttcaccctct gctggctgcc cttcttcatc gttaacattg tgcatgtgat ccaggataac 900

ctcatccgta aggaagttta catcctccta aattggatag gctatgtcaa ttctggtttc 960

aatcccctta tctactgccg gagcccagat ttcaggattg ccttccagga gcttctgtgc 1020

ctgcgcaggt cttctttgaa ggcctatggg aatggctact ccagcaacgg caacacaggg 1080

gagcagagtg gatatcacgt ggaacaggag aaagaaaata aactgctgtg tgaagacctc 1140

ccaggcacgg aagactttgt gggccatcaa ggtactgtgc ctagcgataa cattgattca 1200

caagggagga attgtagtac aaatgactca ctgctgg 1237

<210>5

<211>1110

<212>DNA

<213> Intelligent people

<400>5

ctcatggcgt ccaccacttc cgctgtgcct gggcatccct ctctgcccag cctgcccagc 60

aacagcagcc aggagaggcc actggacacc cgggacccgc tgctagcccg ggcggagctg 120

gcgctgctct ccatagtctt tgtggctgtg gccctgagca atggcctggt gctggcggcc 180

ctagctcggc ggggccggcg gggccactgg gcacccatac acgtcttcat tggccacttg 240

tgcctggccg acctggccgt ggctctgttc caagtgctgc cccagctggc ctggaaggcc 300

accgaccgct tccgtgggcc agatgccctg tgtcgggccg tgaagtatct gcagatggtg 360

ggcatgtatg cctcctccta catgatcctg gccatgacgc tggaccgcca ccgtgccatc 420

tgccgtccca tgctggcgta ccgccatgga agtggggctc actggaaccg gccggtgcta 480

gtggcttggg ccttctcgct ccttctcagc ctgccccagc tcttcatctt cgcccagcgc 540

aacgtggaag gtggcagcgg ggtcactgac tgctgggcct gctttgcgga gccctggggc 600

cgtcgcacct atgtcacctg gattgccctg atggtgttcg tggcacctac cctgggtatc 660

gccgcctgcc aggtgctcat cttccgggag attcatgcca gtctggtgcc agggccatca 720

gagaggcctg gggggcgccg caggggacgc cggacaggca gccccggtga gggagcccac 780

gtgtcagcag ctgtggccaa gactgtgagg atgacgctag tgattgtggt cgtctatgtg 840

ctgtgctggg cacccttctt cctggtgcag ctgtgggccg cgtgggaccc ggaggcacct 900

ctggaagggg cgccctttgt gctactcatg ttgctggcca gcctcaacag ctgcaccaac 960

ccctggatct atgcatcttt cagcagcagc gtgtcctcag agctgcgaag cttgctctgc 1020

tgtgcccggg gacgcacccc acccagcctg ggtccccaag atgagtcctg caccaccgcc 1080

agctcctccc tggccaagga cacttcatcg 1110

<210>6

<211>951

<212>DNA

<213> Artificial

<220>

<223> luciferase

<400>6

gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc 60

ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt 120

ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc 180

ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct 240

tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg 300

ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc 360

gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt 420

gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga 480

ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac 540

aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc 600

atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa 660

ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc 720

gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg 780

acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc 840

ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga 900

aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt g 951

<210>7

<211>696

<212>DNA

<213> Artificial

<220>

<223> luciferase

<400>7

gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc 60

gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct 120

tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt 180

cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc 240

agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca 300

gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg 360

cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa 420

aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt 480

cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt 540

gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct 600

actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat 660

gccagagatc ctatttttgg caatcaaatc attccg 696

<210>8

<211>1363

<212>DNA

<213> Artificial

<220>

<223> luciferase

<400>8

gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc 60

gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct 120

tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt 180

cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc 240

agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca 300

gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg 360

cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa 420

aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt 480

cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt 540

gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct 600

actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat 660

gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt 720

ccattccatc acggttttgg aatgtttact acactcggat atttgatatg tggatttcga 780

gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga ggagccttca ggattacaag 840

attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag cactctgatt 900

gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc cctctctaag 960

gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca aggatatggg 1020

ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg 1080

gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg 1140

ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat gtccggttat 1200

gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct acattctgga 1260

gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct gaagtctctg 1320

attaagtaca aaggctatca ggtggctccc gctgaattgg aat 1363

<210>9

<211>726

<212>DNA

<213> tobacco etch Virus

<400>9

ggagaaagct tgtttaaggg accacgtgat tacaacccga tatcgagcac catttgtcat 60

ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc 120

atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180

catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240

gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300

agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aactaagagc 360

atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg 420

aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 480

gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca 540

agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt 600

ggttggcgat taaatgctga ctcagtattg tgggggggcc ataaagtttt catgagcaaa 660

cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatga attggtgtac 720

tcgcaa 726

<210>10

<211>7

<212>PRT

<213> Artificial

<220>

<223> TEV recognition site

<400>10

Glu Asn Leu Tyr Phe Gln Ser

1 5

<210>11

<211>21

<212>DNA

<213> Artificial

<220>

<223> recognition site of TEV protease

<400>11

gagaacctgt acttccagag c 21

<210>12

<211>7

<212>PRT

<213> Artificial

<220>

<223> recognition site of TEV protease

<400>12

Glu Asn Leu Tyr Phe Gln Val

1 5

<210>13

<211>21

<212>DNA

<213> Artificial

<220>

<223> DNA/V of cleavage site

<400>13

gagaacctgt acttccaggt c 21

<210>14

<211>7

<212>PRT

<213> Artificial

<220>

<223> recognition site

<400>14

Glu Asn Leu Tyr Phe Gln Arg

1 5

<210>15

<211>21

<212>DNA

<213> Artificial

<220>

<223> recognition site

<400>15

gagaacctgt acttccagcg c 21

Claims (20)

1. A method of identifying a compound that modulates a protein-protein interaction between a first protein and a second protein, the method comprising the steps of:

i) providing a first protein linked to a rearranged reporter activating protein, wherein the reporter activating protein comprises a cleavage site for a protease that is interposed between two fragments of the reporter activating protein;

ii) providing a second protein linked to a protease, wherein the protease is capable of cleaving the cleavage site of the reporter activating protein, resulting in the reconstitution of two fragments of the reporter activating protein, thereby activating the reporter activating protein; and wherein association of the first protein with the second protein results in cleavage of the reporter activating protein by the protease;

iii) providing a reporter whose signal is altered by the activity of the reporter activating protein;

iv) providing a test compound;

v) allowing the protease to cleave the cleavage site; and

vi) monitoring reporter signal;

wherein a change in the reporter signal indicates that the protein-protein interaction has occurred.

2. The method of claim 1, wherein at least one of the first protein and the second protein is a membrane bound protein.

3. The method of claim 1, wherein at least one of the first protein and the second protein is a cytoplasmic protein.

4. The method of claim 1, wherein the reporter activating protein is self-activating.

5. The method of claim 1, wherein the reporter activating protein is a reporter.

6. The method of claim 1, wherein the reporter activating protein is an enzyme.

7. The method of claim 1, wherein the reporter activating protein is a protein that causes a change in reporter fluorescence.

8. The method of claim 7, wherein the fluorescent protein is the reporter.

9. The method of claim 1, wherein the first protein forms a fusion protein with a reporter activating protein.

10. The method of claim 1, wherein the protein-protein interaction entails translocation of the first protein or the second protein to a cellular compartment or organelle.

11. The method of claim 1, wherein the method further comprises providing a molecule known to modulate a protein-protein interaction, wherein the test compound modulates the interaction of the molecule with the protein-protein interaction.

12. An assay system comprising:

i) a first protein linked to a rearranged reporter activating protein, wherein the reporter activating protein comprises a cleavage site for a protease that is interposed between two fragments of the reporter activating protein;

ii) a second protein linked to a protease, wherein the protease is capable of cleaving the cleavage site of the reporter activating protein, resulting in the reconstitution of two fragments of the reporter activating protein, thereby activating the reporter activating protein; and

iii) a reporter whose signal is altered by the activity of the reporter activating protein;

wherein association of the first protein with the second protein results in cleavage of the reporter activating protein by a protease.

13. The assay system of claim 12, wherein at least one of the first protein and the second protein is a membrane bound protein.

14. The assay system of claim 12, wherein at least one of said first protein and said second protein is a cytoplasmic protein.

15. The assay system of claim 12, wherein the reporter activating protein is self-activating.

16. The assay system of claim 12, wherein the reporter activating protein is a reporter.

17. The assay system of claim 12, wherein the reporter activating protein is an enzyme.

18. The assay system of claim 12, wherein the reporter activating protein is a protein that causes a change in reporter fluorescence.

19. The assay system of claim 18, wherein the fluorescent protein is the reporter.

20. The assay system of claim 12, wherein the first protein forms a fusion protein with a reporter activating protein.