WO2018143357A1 - Method for screening substance having high affinity for target substance - Google Patents

Abstract

This method for screening a substance having a high affinity for a target substance includes: a step of preparing a measurement sample in which a sample containing a candidate substance is brought into contact with the target substance; a step in which the measurement sample is subjected to imaging mass spectrometry using matrix assisted laser desorption/ionization (MALDI) with the target substance as a matrix, to identify an ionized molecule; and a step of selecting the identified molecule as being a substance having high affinity for the target substance.

Description

Method for screening for substances with high affinity for target substances

The present invention screens a substance having a high affinity with a target substance by using imaging mass spectrometry (Imaging Mass Spectrometry: IMS) using a matrix assisted laser desorption ionization (Matrix Laser Desorption / Ionization: MALDI) method. On how to do.

In drug development, it is important to efficiently select compounds with the expected therapeutic effects from a vast compound library. For this purpose, the simpler the evaluation items, the better. For example, it is preferable to target cells with simpler physiological functions rather than screening for therapeutic effects of candidate compounds on individual animals in which various physiological functions are intricately affected. Furthermore, if the interaction can be analyzed at the level of a molecule directly involved in the therapeutic effect, a compound having the desired therapeutic effect can be selected more efficiently. Reliable evidence, especially for drugs that affect the higher-order functions of the body, such as drugs for treating neuropsychiatric disorders, requires considerable time, investment, and labor to evaluate individually in animal behavioral experiments. Molecular targets based on the above and their evaluation methods are required.

Conventionally, as a method for evaluating the interaction between molecules, a method using a combination of affinity purification such as immunoprecipitation and a detection system such as Western blot or mass spectrometry, an imaging technique such as fluorescence resonance energy transfer (FRET). In general, it is performed by spectrum analysis such as surface plasmon resonance analysis, calorimetric change analysis, high magnetic field nuclear magnetic resonance (NMR), or the like. These methods require high-purity samples, high-quality antibodies, probe production, isotope labeling, and so on, so there are various restrictions in terms of cost, labor, time, safety, etc. To do. For example, in a method that requires a probe for detection, since a specific probe is designed for each observation target, only the interaction between molecules of interest can be measured. In addition, a method that does not use a probe for detection, for example, a method in which a binding molecule for a target substance is detected by liquid chromatography mass spectrometry or the like is generally purified by a method such as immunoprecipitation as a pretreatment. In the process, the detectable molecules are limited. For this reason, a detection system capable of non-target analysis is necessary when conducting research on binding molecules. Furthermore, when there is an extraction process from the living body, the three-dimensional structure may change from that in the living body depending on the purification operation or in vitro environment, and trace molecules that exist only in limited parts of the living body can be recovered in the first place. There are also problems such as difficulty.

Also, in the development of pharmaceuticals, in recent years, development of so-called molecular targeted drugs targeting specific in vivo cells that cause disease, in particular, physiologically active substances that are involved in specific in vivo cells has progressed. This is because the cause of the disease is elucidated at the molecular level or genome level, and the molecular design of the drug (compound having a pharmacological action) targeting the substance related to the disease is performed. Since the therapeutic effect is large and it is difficult to act on in vivo cells other than diseases, an effect of reducing side effects is also expected. On the other hand, it is necessary to (1) elucidate the cause of the disease at the molecular level or genome level, and (2) perform molecular design of a pharmaceutical based on the chemical structure of the identified causative substance. However, both (1) and (2) take a long time to study, and as a result, development has been prolonged.

On the other hand, in recent years, an apparatus for IMS (also referred to as mass microscopy) capable of two-dimensionally visualizing biomolecules on a tissue sample has been developed. In MALDI-based IMS (MALDI-IMS), a sample to be analyzed, such as a tissue slice, is coated with a low-molecular compound (matrix) that absorbs laser light and promotes ionization of biomolecules. Is irradiated with laser light, and an image is reconstructed based on the information of ions detected at each point of the two-dimensional coordinates. In this method, since the tissue section is directly analyzed by two-dimensional laser scanning, the biomolecule on the tissue sample can be ionized while maintaining its position information. The ionized biomolecule is analyzed by a time-of-flight mass spectrometer and identified according to the mass to charge ratio. Thereby, in MALDI-IMS, the distribution of biomolecules on the tissue sample can be imaged by the relative value of the signal intensity between the measurement points. As the matrix, a compound suitable for ionization of biomolecules has been used empirically. For example, an ion-dissociable substance having a molecular weight of 50 or more and less than 300, such as 3,5-methoxy-4-hydroxycinnamic acid and 9-aminoacridine (9AA) is used, and a matrix ionized with biomolecules on a tissue sample The molecules are desorbed and fly from the tissue sample in a bound state, and are separated and identified for each mass-to-charge ratio (m / z) by the analyzer in MALDI-IMS.

For example, Patent Document 1 discloses a method for evaluating the therapeutic effect of a candidate compound for a breast cancer therapeutic agent using MALDI-IMS. In this method, since a large amount of phosphatidylcholine (PC) of a specific molecular species is present in breast tissue due to abnormal lipid metabolism in breast cancer, the molecule of phosphatidylcholine in the breast tissue of a subject administered with a candidate compound The abundance ratio of the species is measured by MALDI-IMS, and when the abundance ratio of molecular species present in a large amount in breast cancer patients is low, the candidate compound is evaluated to be effective as a breast cancer therapeutic agent. In other words, this method involves directly administering a candidate compound to a subject and comparing before and after exhibiting an anticancer effect through metabolism, so the types of candidate substances that can be used for screening are naturally limited. There was a limit to the efficiency of screening.
In addition, conventionally, no method has been found to measure the affinity involving a substance in a living body as well as the above-described intermolecular interaction using MALDI-IMS.

JP 2014-149243 A

An object of the present invention is to provide a method for screening a substance having high affinity with a target substance using MALDI-IMS.

The present invention provides a method for screening a substance having a high affinity with the following target substance.
[1] preparing a measurement sample in which a target substance and a sample containing a candidate substance are brought into contact;
Performing an imaging mass spectrometry method using the MALDI method with the target substance as a matrix on the measurement sample, and identifying ionized molecules;
Selecting the identified molecule as a substance having high affinity with the target substance;
A method for screening a substance having high affinity with a target substance.
[2] The step of preparing the measurement sample is as follows:
Contacting a test sample containing a candidate substance with a target substance;
Drying the contacted sample and the target substance in a contact state to obtain a measurement sample, and
The identification step performs a spectrum scanning step of obtaining an ionized molecule spectrum by performing imaging mass spectrometry using MALDI method on the measurement sample,
Identifying a peak substance from the spectrum,
The method of [1].
[3] The method according to [1] or [2] above, wherein the target substance is a compound having a molecular weight of 10,000 or less.
[4] The method according to any one of [1] to [3], wherein the candidate substance is a phospholipid.
[5] The samples according to [1] to [4], wherein the sample containing the candidate substance is a tissue section, and the measurement sample is obtained by attaching the target substance to the surface of the sample containing the candidate substance. Either way.
[6] The wavelength of a laser used for imaging mass spectrometry using the MALDI method is 200 to 400 nm,
The method according to any one of [1] to [5], wherein the target substance is a compound having a molecular weight of 300 to 10,000 containing an aromatic ring and / or a heterocyclic ring.
[7] Using a substance different from the target substance as a control substance,
Preparing a control sample for measurement in which the sample containing the control substance and the candidate substance is contacted;
Performing an imaging mass spectrometry method using the MALDI method with the control substance as a matrix on the measurement control sample, and identifying ionized molecules;
Selecting the identified molecule as a substance having a high affinity with the control substance,
Of the molecules ionized from the measurement sample, select a molecule other than a molecule selected as a substance having a high affinity with the control substance as a substance having a high affinity with the target substance.
Any one of [1] to [6] above.
[8] The control substance is 3,5-methoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, trans-4-hydroxy-3-methoxycinnamic acid, 2,5-dihydroxy The method according to [7] above, which is benzoic acid, 3-hydroxypicolinic acid, 9-aminoacridine, 2,5-dihydroxyacetophenone, or 1,8-dihydroxy-9,10-dihydroanthracen-9-one.
[9] A step of bringing a test sample containing a target substance into contact with a candidate substance;
Drying the test sample and the candidate substance in a contact state to obtain a measurement sample;
A scanning step of performing imaging mass spectrometry using MALDI method on the measurement sample and scanning m / z corresponding to the target substance among the ionized molecules;
A peak intensity measuring step for measuring the peak intensity at the m / z;
Selecting the candidate substance in which the peak intensity is detected as a substance having a high affinity with the target substance.
A method for screening a substance having high affinity with a target substance.
[10] The method according to [9] above, wherein the candidate substance is a compound having a molecular weight of 10,000 or less.
[11] The method according to [9] or [10] above, wherein the target substance is a phospholipid.
[12] Any of [9] to [11], wherein the sample containing the target substance is a tissue section, and the measurement sample is obtained by attaching the candidate substance to the surface of the sample containing the target substance. That way.
[13] The wavelength of a laser used for imaging mass spectrometry using the MALDI method is 200 to 400 nm, and the candidate substance is a compound having a molecular weight of 300 to 10,000 including an aromatic ring and / or a heterocyclic ring. 9] to any one of [12].
[14] A method for designing a medicine comprising a screening step for a substance having a high affinity with the target substance of any one of [9] to [13], and a candidate substance selected by the screening process as an active ingredient .
[15] A method for producing a medicine, comprising a screening step of a substance having a high affinity with the target substance of any one of [9] to [13], and containing a candidate substance selected by the screening process as an active ingredient .

By the method for screening candidate substances having high affinity with the target substance according to the present invention, candidate substances having strong affinity for the target substance can be comprehensively selected without limiting the molecules to be analyzed. For this reason, the method is particularly suitable for selecting a substance (biomolecule) having high affinity for a target substance of interest from a sample containing various biomolecules such as a biological sample such as a tissue slice.
In addition, the method for screening candidate substances having high affinity with the target substance (biomolecule) according to the present invention comprehensively selects candidate substances having strong affinity for the target substance without limiting the molecules to be analyzed. can do. For this reason, this method is particularly suitable for selecting substances exhibiting high affinity for the target substance (biomolecule) of interest from various candidate compounds that can have a pharmacological action.

It is process drawing which shows one Embodiment of this invention. In Example 1, it is the mass spectrum acquired by MALDI-IMS performed with respect to the mouse | mouth brain slice. In Example 1, it is the molecular distribution image figure which built two-dimensionally the spectrum data acquired by MALDI-IMS according to ionic strength. In Example 1, it is the result of the multistage MS analysis with respect to the ion peak characteristic of clozapine. In Example 1, it is the mass spectrum acquired by MALDI-IMS performed with respect to PIP2 spotted on the electroconductive slide glass. In Example 1, it is the figure which showed the result of the liposome binding experiment. It is process drawing which shows other embodiment of this invention.

The method for screening a substance having high affinity with the target substance according to the present invention (hereinafter sometimes referred to as “the screening method according to the present invention”) is a screening method using MALDI-IMS.

It has been suggested that co-crystal formation of appropriate matrix molecules and biomolecules is important for ionization as the MALDI-IMS technique is developed and developed. Therefore, it has been considered that the formation of a dense co-crystal between the matrix molecule and the biomolecule is important for the ionization of the biomolecule efficiently. Based on this idea, when observing the in-plane distribution of a specific biomolecule in a section of a biological sample, as described above, 3,5-methoxy-4-hydroxycinnamic acid, 9-amino is used as a matrix. An ion dissociable substance having a molecular weight of 50 or more and less than 300, such as acridine (9AA), has been used. These have been used by many researchers for many years from the viewpoint of forming a co-crystal and easily obtaining a clear two-dimensional image.

Specifically, the screening method according to the present invention includes a step of preparing a measurement sample in which a target substance and a sample containing a candidate substance (test sample) are brought into contact, and the target for the measurement sample. MALDI-IMS using a substance as a matrix to identify an ionized molecule, and selecting the identified molecule as a substance having a high affinity for the target substance. When MALDI-IMS is performed using the target substance as a matrix, a substance having a high affinity with the target substance in the sample is ionized. Therefore, by identifying this ionized molecule and selecting it as a substance with high affinity for the target substance, it is possible to comprehensively select substances that have a strong affinity for the target substance contained in the sample. it can.

Among the steps described above, the step of preparing the measurement sample includes the step of bringing the test sample and the prepared target substance into contact with each other, drying the contacted sample and the target substance in a contact state, and measuring the sample. And a drying step as a sample. In addition, the identification step includes a spectrum scanning step for obtaining a spectrum of ionized molecules by performing imaging mass spectrometry using a MALDI method on the measurement sample, and an identification step for identifying a peak substance from the spectrum. Contains.

Hereinafter, the screening method of the present invention will be described in detail with reference to FIG. Here, it demonstrates as an example which searches for a candidate substance with high affinity in the living body with respect to a known target substance. Here, “high affinity” is used synonymously with “having affinity”. Further, the test sample preparation process and the target substance preparation process will be described.

FIG. 1 is a process diagram showing a screening method of the present invention. A test sample preparation process 100, a target substance preparation process 200, a contact process 300, a drying process 500, and an imaging mass spectrometry method using a MALDI method. (MALDI-IMS) spectrum scanning step 600 and peak substance identifying step 900 are included.

The test sample creation step 100 is a test sample creation step for creating a test sample containing a candidate substance.
In the present invention, a candidate substance, that is, a substance expected to be a substance having a high affinity with a target substance described later is not particularly limited as long as it is a substance ionized by MALDI-IMS. Examples include organic compounds (also referred to as biomolecules) contained in living bodies such as sugars, nucleic acids, proteins, lipids, organic acids, and vitamins. In the present invention, identification of molecules ionized in IMS is easier, so that the substance has a relatively small molecular weight, such as phospholipid, peptide, oligonucleotide, vitamin, and the like, and is relatively easy to identify in IMS. Is preferred. Among them, the candidate substance is preferably a phospholipid or an organic acid, and more preferably a phospholipid, from the point that the analysis results by MALDI-IMS are substantial. Examples of phospholipids include phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP, PIP1), phosphatidylinositol diphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3), phosphatidylcholine (PC), phosphatidylethanolamine Examples thereof include glycerophospholipids such as (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and diphosphatidylglycerol (cardiolipin), and sphingophospholipids such as sphingomyelin.

In the present invention, the test sample is not particularly limited as long as it contains a candidate substance. For example, the test sample may be a sample collected from a living body, or an artificially prepared sample. Also good. Examples of biological samples include tissue fragments collected from living organisms, blood, lymph, bone marrow, ascites, exudates, amniotic fluid, sputum, saliva, semen, bile, pancreatic juice, urine, and other body fluids, intestinal lavage fluid, lungs An organ cleaning solution such as a cleaning solution may be used. Examples of the artificially prepared sample include purified known substances or a mixture of two or more kinds of purified known substances.

When using a body fluid, a washing solution, or the like as a test sample, it is also preferable to concentrate and purify candidate substances in advance by centrifugation, chromatography, electrophoresis, or the like. The tissue piece may be used as a lump, or a suspension obtained by homogenizing the tissue piece in an appropriate buffer may be used as a test sample. Collection of tissue pieces and body fluids from a living body, washing solution, centrifugation, chromatography, electrophoresis, homogenization, and the like can be performed by conventional methods.

As a test sample in the present invention, a tissue piece collected from a living body is preferable because it is possible to examine the affinity with a target substance in a state in which the environment in the living body is relatively maintained by utilizing the characteristics of IMS. A tissue section in which a tissue piece is thinned is more preferable. In the present invention, the “tissue piece” includes not only a tissue piece itself collected from a living body but also a tissue piece embedded in paraffin and a tissue piece deparaffinized after embedding in paraffin. Similarly, in the present invention, the “tissue section” means a section of a tissue piece obtained by embedding a tissue piece collected from a living body in addition to a frozen section of a tissue piece collected from a living body, Also included are sections of paraffin tissue pieces. In addition, collection | recovery of the tissue piece from a biological body, paraffin embedding, and deparaffinization can be performed by a conventional method.
Further, the tissue piece collected from the living body may be collected from a healthy living body, or may be collected from a living body having a disease or a disorder. For example, a so-called pathological tissue may be used.

Next, the target substance preparation step 200 is a target substance preparation step for preparing a target substance. In the present invention, the target substance is a target substance for screening for a substance (candidate substance) having high affinity with the substance. The target substance is not particularly limited as long as it can function as a matrix in MALDI-IMS. For example, sugars, nucleic acids as well as artificial compounds such as pharmaceuticals and low molecular compounds (chemical libraries, etc.) , Organic compounds contained in living bodies such as proteins, lipids, organic acids and vitamins. In the present invention, an organic compound having a molecular weight of 10,000 or less is preferable because it is easily excited by selecting a laser beam having an appropriate wavelength in IMS and is suitable as a matrix. However, the molecular weight is more than 10,000. The organic compound may be used. The molecular weight of the organic compound is typically 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less, and 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more.

Also, MALDI-IMS differs in the chemical structure of a compound suitable for screening a substance having a high affinity because the excited energy differs depending on the laser wavelength used. Specifically, in the case of MALDI-IMS using a laser that oscillates at a wavelength of 200 to 400 nm, if the compound is an organic compound having an aromatic ring and / or a heterocyclic ring, the energy of the laser is efficiently converted, and Candidate substances (biomolecules) can be ionized together.

As a method for preparing the target substance, it is desirable to dissolve or suspend it in a solution. The solution for suspending or dissolving the target substance is not particularly limited as long as it is a solution that can be suspended or dissolved without chemically altering the target substance. Phosphate buffer, Tris buffer, etc. These can be selected appropriately from various buffers, organic solvents such as alcohols and ketones, or water according to the target substance. In the present invention, alcohols such as methanol and ethanol are preferable because they have little influence on a biological sample or an organic compound contained in the living body and easily volatilize.
Moreover, when the contact process 300 mentioned later is vapor deposition, it may remain a powder or paste-like solid.

Next, the contact process 300 is a contact process in which the prepared test sample is brought into contact with the prepared target substance. Specifically, in the present invention, first, as a measurement sample to be subjected to MALDI-IMS, the target substance prepared in the target substance preparation step 200 and the test sample prepared in the test sample preparation step 100 were brought into contact with each other. Prepare one. The measurement sample is not particularly limited as long as the candidate substance in the test sample is in contact with the target substance. However, the measurement sample is preferably flat so that measurement by IMS is possible. As a method for attaching the target substance to the surface of the test sample, for example, a method in which a solution in which the target substance is dissolved or suspended in a buffer is sprayed so as to uniformly adhere to the surface of the test substance, the solution is used. Examples thereof include a method of applying with a brush or the like, a method of immersing a test sample in the solution, and the like.
For example, when the test sample is a suspension in which candidate substances are dispersed, a mixture obtained by mixing the test sample and the target substance is a thin film such as a PVDF (polyvinylidene fluoride) film or a nitrocellulose film. Or a sample plotted on a conductive slide glass can be used as a measurement sample. In addition, a solution in which a candidate substance is dispersed or dissolved is plotted on a thin film such as a PVDF film or a nitrocellulose film or a conductive slide glass, and the target substance is attached to the surface of this thin film. can do. When the test sample is a tissue piece or a tissue section, a sample having a target substance attached to these surfaces can be used as a measurement sample.
Further, the contact process 300 may be a vapor deposition method such as vacuum deposition.

Next, the drying process 500 is a drying process in which the contacted sample and the target substance are dried in a contact state to obtain a measurement sample. The mass spectrometer used for the MALDI-IMS method is maintained in a high vacuum state. Immediately after the contact process 300, volatile components (solvents, etc.) in the solution may be contained, and if it is directly subjected to MALDI-IMS, gas is generated in the apparatus and the degree of vacuum necessary for analysis is maintained. There is a risk of not being able to hit. For this reason, drying is performed for a predetermined time according to the state of the measurement sample. As an example, air drying can be performed for about 1 to 120 minutes.
At this time, it is desirable that the contact process 400 and the drying process 500 are continuous, and it is desirable to shift to a process while maintaining the state without tilting the sample. For example, in the case of a flat sample such as a tissue section, it is desirable to perform the contact process 400 in a horizontal state, and then proceed to the drying process 500 continuously while maintaining the horizontal state.

The drying process 500 may use heating means such as a hot plate or an infrared lamp in order to shorten the time. However, it is necessary to pay attention to the heating temperature so that the test sample is not thermally decomposed and migration of candidate substances in the test sample does not occur. Further, the drying process 500 is not limited to the atmosphere, and may be performed in a vacuum apparatus different from the MALDI-IMS. In this case, even if gas is generated in the drying process 500, it is only necessary to lower the vacuum degree of the vacuum device, and there is an advantage that the MALDI-IMS device is not contaminated.

Note that a co-crystal growth step 400 (not shown) may be provided between the contact step 300 and the drying step 500. The co-crystal growth process 400 is held at a predetermined temperature, a predetermined humidity or a spatial solvent concentration, and a predetermined time so that the co-crystal is formed and grows when the target substance and the candidate substance can form a co-crystal. For example, it is left in a closed space such as a petri dish or a desiccator.

Next, MALDI-IMS is performed on the prepared measurement sample, and ionized molecules are identified. Specific processes include a spectrum scan process 600 and a peak substance identification process 800.

Of these, the spectrum scan step 600 is a step of performing a spectrum scan in a predetermined m / z range by an imaging mass spectrometry method (MALDI-IMS) using the MALDI method. One example is a scan in the range of mass-to-charge ratio (m / z) from 0 to 3000, and another example is a scan in the range of 500 to 2500 m / z. In order to detect a candidate substance without omission, it is desirable to set a wide scan range. However, when a candidate substance can be predicted to some extent, a scan with a relatively narrow range including an expected m / z may be used. Here, MALDI-IMS can be implemented by a conventional method. For example, a test sample obtained through the drying process 500 and having a target substance attached to the surface thereof is placed on a glass slide coated with ITO (indium oxide), and this glass slide is placed in an imaging mass spectrometer. Then, the organic compound in the test sample is ionized while maintaining the position information by two-dimensional laser scanning, and a mass spectrum is acquired.

The step 800 for identifying the peak material is a step for identifying the peak material from a spectrum in a predetermined range obtained from MALDI-IMS. For example, mass spectra acquired using software attached to the apparatus or commercially available software are analyzed, and ionized molecules corresponding to the peaks are identified. For identification of molecules from mass spectra, refer to known databases (eg (http://www.lipidmaps.org/data/structure/LMSDSearch.php)) according to mass-to-charge ratio (m / z) Can be done. In this way, a candidate substance (biomolecule) having a high affinity for the target substance in vivo can be identified.
The identified molecule can be confirmed to be able to obtain the same mass spectrum by performing MALDI-IMS in the same manner using the target substance alone.

Note that the mass spectrum acquired by MALDI-IMS may include a mass spectrum of molecules that are not highly compatible with the target substance but are easily ionized. Therefore, in order to specify a mass spectrum of a substance having a high affinity for the target substance, MALDI-IMS is similarly performed on the same test sample using a substance different from the target substance as a control substance (Steps 100, 200, 300). , 500, 600, and 800), by removing the mass spectrum obtained using the control substance as a matrix as noise, the accuracy of screening can be improved. Specifically, a step of preparing a control sample for measurement in which a control substance and a sample containing the candidate substance are brought into contact with each other, and MALDI-IMS using the control substance as a matrix for the control sample for measurement are prepared. Performing the step of identifying the ionized molecule and the step of selecting the identified molecule as a substance having a high affinity with the control substance, and among the molecules ionized from the measurement sample, A molecule other than the molecule selected as a substance having a high affinity with the control substance is selected as a substance having a high affinity with the target substance.

The control substance is not particularly limited as long as it is a substance other than the target substance. For example, a compound widely used as a matrix in MALDI-IMS can be used because excitation by laser irradiation is easy. Examples of the general-purpose compound include 3,5-methoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, trans-4-hydroxy-3-methoxycinnamic acid, and 2,5-dihydroxy. Examples include benzoic acid, 3-hydroxypicolinic acid, 9-aminoacridine (9AA), 2,5-dihydroxyacetophenone, or 1,8-dihydroxy-9,10-dihydroanthracen-9-one. These general-purpose compounds are not drugs, and the peak of mass spectrum obtained by MALDI-IMS can be treated as negative data (noise).

In addition, in order to screen for a substance that specifically binds to the target substance, a substance having a structure similar to that of the target substance can be used as a control substance. In this case, the peak of the mass spectrum obtained by MALDI-IMS can be handled as positive data supporting the peak of the candidate substance obtained for the target substance.

In addition, for example, by performing the screening method according to the present invention using a target substance as a drug having some physiological function and a tissue section as a test sample, the target substance can be specifically identified with the drug in an environment closer to the living body. It is possible to exhaustively search for biomolecules that can bind. In addition, it is possible to confirm from which part of the living tissue the spectrum data obtained by MALDI-IMS is two-dimensionally constructed according to the ion intensity. Information on the localization of a molecule that binds to a drug in a tissue is useful for elucidating the mechanism of action of the drug in vivo. In particular, in the present invention, it is possible to analyze by focusing on a site important for the pharmacological effect by appropriately selecting a tissue section, and it is possible to analyze a tissue section having a thickness of about 10 μm at intervals of 5 μm in length and width. A trace amount of biomolecules present only at a local site can be analyzed without prior purification.

Further, the characteristic ion peak can be analyzed in detail by performing a multi-stage MS analysis process 700 (not shown) subsequent to the spectrum scan process 600. For this reason, the screening method according to the present invention can be a powerful means for exhaustively searching for biomolecules having affinity for pharmaceutical products and candidate compounds.

Since the screening method according to the present invention detects and identifies a substance having a high affinity with a target substance by mass spectrometry, it is not necessary to develop a high-quality probe for each target substance in order to detect an interaction, and it is comprehensive. Exploratory research is possible. In addition, since a tissue section can be used as a test sample as it is, biomolecules (candidate substances) are held in a state close to the living body, so that it is difficult to maintain the original structure by extraction operation. Suitable for analysis of constituent molecules. In addition, since it is not necessary to extract biomolecules from tissue sections, screening can be performed more rapidly.

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
An example of searching for a biomolecule (candidate substance) showing affinity for a specific drug (target substance) will be described below.
Among antipsychotic drugs, biomolecules (candidate substances, affinity molecules) that specifically bind to clozapine (target substance) were comprehensively searched from brain tissue.
First, a mouse brain section was prepared (test sample preparation step 100). A clozapine (Clz) solution (2 mg / mL) in which clozapine (CAS No. 5786-21-0, molecular weight: 327) was dissolved in methanol was prepared (target substance preparation step 200). Subsequently, the clozapine solution was spray applied to the mouse brain slice by spraying and contacted (contact process 300). Subsequently, crystals were formed on the tissue surface by natural drying (co-crystal growth process 400 and drying process 500). Next, this section was placed on an ITO-coated slide glass, and this slide glass was placed in a MALDI-IMS imaging mass spectrometer (“Solarix XR”, Bruker Daltonics) to perform two-dimensional laser scanning. (YAG laser, wavelength 355 nm) The organic compound in the test sample was ionized while maintaining its positional information, and mass spectra were acquired for m / z of 600 to 1600 (spectrum scanning process 600).

Subsequently, as a control, instead of clozapine, MALDI-IMS was similarly performed using 9-aminoacridine (9AA) (2 mg / mL methanol solution) or olanzapine (Olz) (2 mg / mL methanol solution). Mass spectra were acquired. Note that 9-aminoacridine (9AA) is an example of a compound that does not have an affinity for the aforementioned biomolecule. Olanzapine (Olz) is one of antipsychotic drugs, like clozapine, but has a different molecular structure from clozapine and has a different pharmacological action.

The acquired mass spectrum is shown in FIG. In FIG. 2, (a) “9AA” is a mass spectrum obtained using 9-aminoacridine, and FIG. 2 (b) “Clz” is a mass spectrum obtained using clozapine. ) “Olz” is a mass spectrum acquired using olanzapine. In FIGS. 2A to 2C, common peaks are observed in the vicinity of m / z 888 and 905. FIG. On the other hand, with respect to clozapine, which is an antipsychotic drug, specific peaks not observed elsewhere are observed at m / z 965 and 1045 (FIG. 2B).

Next, FIG. 3 shows a molecular distribution image diagram in which the obtained spectrum data is two-dimensionally constructed according to the ionic strength (m / z is four conditions of 885.55, 888.62, 965.52, 1045.48). ). In the figure, the meanings of the abbreviations “9AA”, “Clz”, and “Olz” are the same as described above. “9AA”, “Clz”, and “Olz” have different molecular distribution images, and the distribution in the mouse brain of molecules with high affinity for clozapine and molecules with high affinity for olanzapine is different. It was confirmed that For clozapine, clear images were obtained at all m / z including m / z 965.52 and 1045.48, and this result was in good correspondence with the previous mass spectrum (Fig. 2 (b)). Is shown.

From the mass spectrum of FIG. 2 (b), an ion peak characteristic of clozapine, that is, an ion peak that is not detected when 9-aminoacridine or olanzapine is used among ion peaks detected when clozapine is used. (M / z 965 and 1045) were selected as ion peaks of molecules having high affinity for clozapine. When these ion peaks were subjected to multistage MS analysis (multistage MS process 700), they were identified as PIP1 and PIP2. FIG. 4 shows the results of multi-stage MS analysis for ion peaks characteristic of clozapine. FIG. 4 (a) shows only the m / z 965 ion obtained in the process of the spectrum scan process 600, and further decomposes it by giving kinetic energy as multi-stage MS analysis (MS / MS). Is observed. FIG. 4B shows only the m / z 1045 ions obtained in the process of the spectrum scanning process 600, which is further decomposed by giving kinetic energy as multi-stage MS analysis (MS / MS). The spectrum was observed. From the peak m / z of the fragment ions of each decomposed product, the ion of m / z 965 was identified as PIP1, and the ion of m / z 1045 was identified as PIP2. From these results, PIP1 and PIP2 could be selected as molecules having high affinity with clozapine. On the other hand, olanzapine showed no affinity with PIP1 and PIP2, suggesting that the difference in pharmacological action between clozapine and olanzapine as an antipsychotic is due to the difference in affinity between PIP1 and PIP2.

Next, of PIP1 and PIP2, the identification result was confirmed on behalf of PIP2. Specifically, instead of the mouse brain slice, Phosphatidylinositol 4,5-bisphosphate diC4 (PI (4,5) P2 diC4) (molecular weight 744, Echelon Biosciences Inc. catalog number P-4504), which is a standard substance of PIP2, is used. Used as a test sample. Specifically, mass spectrometry is performed by performing MALDI-IMS in the same manner except that P-4504 spotted on a conductive slide glass is used as a test sample and a methanol solution of clozapine or olanzapine is mixed on the glass. did. As a result, a strong ion peak at m / z 745 was detected from the spot mixed with clozapine, but only a very weak ion peak at m / z 745 was detected from the spot mixed with olanzapine or 9-aminoacridine. There wasn't. A mass spectrum acquired from a spot mixed with clozapine is shown in FIG. 5A, and a mass spectrum acquired from a spot mixed with olanzapine is shown in FIG. 5B. In the figure, the meanings of the abbreviations “Clz” and “Olz” are the same as described above.

Furthermore, the lipid binding ratio in vitro was examined for PIP2. Specifically, a liposome (control liposome) containing PC and PE at PC: PE = 80: 20 (molar ratio), and PC, PE and PIP2 are PC: PE: PIP2 = 80: 18: 2 (molar ratio). ) Was prepared, and a liposome binding experiment for clozapine and olanzapine was performed using the liposome. Specifically, first, 100 μM liposomes and 10 μM clozapine or olanzapine were incubated in HEPES buffer, and then ultracentrifuged at 300,000 × g, and separated into supernatant and precipitate. The resulting supernatant and precipitate were redissolved with a buffer solution, respectively, and the absorbance at a wavelength of 224 nm was measured. The ratio of clozapine or olanzapine contained in the precipitate out of total clozapine or olanzapine as the lipid binding ratio. Calculated. The results are shown in FIG. In the figure, “Clz” is the result of the binding experiment for clozapine, and “Olz” is the result of the binding experiment for olanzapine. As a result, it was confirmed that clozapine was bound to PIP2 liposome and olanzapine was not bound to PIP2 liposome.
That is, the candidate substance PIP2 selected for the target substance clozapine by the screening method of the present invention showed high affinity that can be said to be specific for the target substance clozapine in the buffer solution. From this, it was proved that the screening method of the present invention is effective in determining the presence or absence of affinity between two substances in vivo.

In addition, it is clear from the above demonstration that it is possible in principle to search for a compound having a high affinity with the target substance as a candidate substance using the target substance as a biomolecule.

Hereinafter, the second screening method of the present invention will be described in detail with reference to FIG. Here, it demonstrates as an example which searches for a candidate substance with high affinity in the living body with respect to the known standard substance (biomolecule) in the living body. Further, the test sample preparation process and the target substance preparation process will be described.

FIG. 7 is a process diagram showing a second screening method of the present invention, in which a test sample preparation process 150, a candidate substance preparation process 250, a contact process 300, a drying process 500, and an imaging using a MALDI method are performed. A target substance (m / z) scanning step 650 by mass spectrometry (MALDI-IMS) and a peak intensity measuring step 900 are included.

The test sample creation step 150 is a test sample creation step for creating a test sample containing a target substance.

In the present invention, the target substance, that is, a substance expected to be a substance having a high affinity with a candidate substance described later is not particularly limited as long as it is a substance ionized by MALDI-IMS. Examples include organic compounds (also referred to as biomolecules) contained in living bodies such as sugars, nucleic acids, proteins, lipids, organic acids, and vitamins. In the present invention, identification of molecules ionized in IMS is easier, so that the substance has a relatively small molecular weight, such as phospholipid, peptide, oligonucleotide, vitamin, and the like, and is relatively easy to identify in IMS. Is preferred. Among them, the candidate substance is preferably a phospholipid or an organic acid, and more preferably a phospholipid, from the point that the analysis results by MALDI-IMS are substantial. Examples of phospholipids include phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP, PIP1), phosphatidylinositol diphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3), phosphatidylcholine (PC), phosphatidylethanolamine Examples thereof include glycerophospholipids such as (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and diphosphatidylglycerol (cardiolipin), and sphingophospholipids such as sphingomyelin.

In the present invention, the test sample is not particularly limited as long as it contains the target substance. For example, the test sample may be a sample collected from a living body, or an artificially prepared sample. Also good. Examples of biological samples include tissue fragments collected from living organisms, blood, lymph, bone marrow, ascites, exudates, amniotic fluid, sputum, saliva, semen, bile, pancreatic juice, urine, and other body fluids, intestinal lavage fluid, lungs An organ cleaning solution such as a cleaning solution may be used. Examples of the artificially prepared sample include purified known substances or a mixture of two or more kinds of purified known substances.

When using a body fluid, a washing solution, or the like as a test sample, it is also preferable to concentrate and purify candidate substances in advance by centrifugation, chromatography, electrophoresis, or the like. The tissue piece may be used as a lump, or a suspension obtained by homogenizing the tissue piece in an appropriate buffer may be used as a test sample. Collection of tissue pieces and body fluids from a living body, washing solution, centrifugation, chromatography, electrophoresis, homogenization, and the like can be performed by conventional methods.

As a test sample in the present invention, taking advantage of the characteristics of IMS, it is possible to examine the affinity with a candidate substance in a state in which the environment in the living body is relatively maintained, a tissue piece collected from a living body is preferable, A tissue section in which a tissue piece is thinned is more preferable. In the present invention, the “tissue piece” includes not only a tissue piece itself collected from a living body but also a tissue piece embedded in paraffin and a tissue piece deparaffinized after embedding in paraffin. Similarly, in the present invention, the “tissue section” means a section of a tissue piece obtained by embedding a tissue piece collected from a living body in addition to a frozen section of a tissue piece collected from a living body, Also included are sections of paraffin tissue pieces. In addition, collection | recovery of the tissue piece from a biological body, paraffin embedding, and deparaffinization can be performed by a conventional method.
Further, the tissue piece collected from the living body may be collected from a healthy living body, or may be collected from a living body having a disease or a disorder. For example, a so-called pathological tissue may be used.

Next, the candidate substance preparation step 250 is a candidate substance preparation step for preparing a candidate substance. In the present invention, the candidate substance is a substance intended for screening a substance having a high affinity for the target substance. Candidate substances are not particularly limited as long as they can function as a matrix in MALDI-IMS. For example, artificial substances such as pharmaceuticals and low-molecular compounds (chemical libraries, etc.), sugars, nucleic acids, etc. , Organic compounds contained in living bodies such as proteins, lipids, organic acids and vitamins. In the present invention, an organic compound having a molecular weight of 10,000 or less is preferable because it is easily excited by selecting a laser beam having an appropriate wavelength in IMS and is suitable as a matrix. However, the molecular weight is more than 10,000. The organic compound may be used. The molecular weight of the organic compound is typically 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less, and 100 or more, 200 or more, 300 or more, 400 or more, or 500 or more.

Also, MALDI-IMS differs in the chemical structure of a compound suitable for screening a substance having a high affinity because the excited energy differs depending on the laser wavelength used. Specifically, in the case of MALDI-IMS using a laser that oscillates at a wavelength of 200 to 400 nm, if the compound is an organic compound having at least one aromatic ring and / or heterocyclic ring, the energy of the laser is efficiently converted. Both the compound and the target substance (biomolecule) can be ionized.

As a method for preparing a candidate substance, it is desirable to dissolve or suspend it in a solution. The solution for suspending or dissolving the candidate substance is not particularly limited as long as it is a solution that can suspend or dissolve the candidate substance without chemically altering the candidate substance. Phosphate buffer, Tris buffer, etc. These can be selected appropriately from various buffers, organic solvents such as alcohols and ketones, or water according to the target substance. In the present invention, alcohols such as methanol and ethanol are preferable because they have little influence on a biological sample or an organic compound contained in the living body and easily volatilize.
Moreover, when the contact process 300 mentioned later is vapor deposition, it may remain a powder or paste-like solid.

Next, the contact process 300 is a contact process in which the prepared test sample is brought into contact with the prepared target substance, and is the same as the contact process 300 described in the first screening method of the present invention.

Next, the drying process 500 is a drying process in which the contacted sample and the target substance are dried in a contact state to obtain a measurement sample, which is the same as the drying process 500 described in the first screening method of the present invention. It is.

Also, the co-crystal growth process 400 (not shown) is the same as the co-crystal growth process 400 described in the first screening method of the present invention.

Next, MALDI-IMS is performed on the prepared measurement sample, and ionized molecules are identified. Specific steps include a target substance (m / z) scanning step 650 and a step 900 of measuring peak intensity.

Among these, the target substance (m / z) scanning step 650 is a step of scanning m / z corresponding to the target substance (biomolecule) by imaging mass spectrometry (MALDI-IMS) using the MALDI method. (For example, in Example 1, when PIP2 is the target substance, m / z 1045 or a peripheral region including m / z 1045 (for example, a range within ± 5% with respect to the center m / z) Scan about). Here, MALDI-IMS can be implemented by a conventional method. For example, a test sample obtained by passing through the drying step 500 and having a target substance attached to the surface thereof is placed on a glass slide coated with ITO (indium oxide), and the glass slide is connected to a MALDI-IMS imaging mass spectrometer. The organic compound in the test sample is ionized while maintaining the position information by two-dimensional laser scanning, and a mass spectrum is acquired.

The step 900 of measuring the peak intensity is to measure the peak intensity at the corresponding m / z of the target substance (biomolecule), and to quantify it if necessary. This measurement process 900 includes a process of determining the presence or absence of a peak. As a first method of determination, if there is a peak at m / z 1045, there is a peak if there is a significant difference in peak intensity compared to the noise area around the peak (for example, m / z 1200) It is determined. In addition, as a second method for determining, when there is a peak at m / z 1045, the peak intensity is compared with the peak at m / z 1045 of the reference substance spectrum (negative data) such as 9AA described above. If a significant difference is recognized, it is determined that there is a peak. As a third method for determining, when there is a peak at m / z 1045, the peak intensity is digitized and stored in a memory or the like, and a peak value at m / z 1045 for a plurality of candidate substances. By comparing with the above, the peak with the largest peak value is determined as having a peak (or peak maximum).

Thus, for example, a section of tissue such as a pathological tissue or a disease model mouse is used as a test sample, a biomolecule related to a disease is used as a target substance, and a compound that can be expected to have a pharmacological effect is used as a candidate substance. By performing the screening method, candidate drugs that can specifically bind to the biomolecule in an environment closer to the living body can be exhaustively searched. In addition, it is possible to confirm from which part of the living tissue the spectrum data obtained by MALDI-IMS is two-dimensionally constructed according to the ion intensity. Information on the localization of a molecule that binds to a drug in a tissue is useful for confirming the mechanism of action of the drug in vivo, and information on side effects can be obtained from the distribution state in a normal tissue. . In particular, in the present invention, it is possible to analyze by focusing on a site important for the pharmacological effect by appropriately selecting a tissue section, and it is possible to analyze a tissue section having a thickness of about 10 μm at intervals of 5 μm in length and width. A trace amount of biomolecules present only at a local site can be analyzed without prior purification. Further, the characteristic ion peak can be structurally analyzed by performing a multi-stage MS analysis process 700 (not shown) following the spectrum scan process 600. For this reason, the screening method according to the present invention can be a powerful means for comprehensively searching for drugs and candidate compounds having affinity for biomolecules.

Also, depending on the disease, a plurality of biomolecules may be involved in the disease independently or in cooperation with each other. As an application of the present invention, it is possible to set a plurality of target substances (biomolecules) and perform a two-dimensional search for a plurality of candidate substances (compounds that can be expected to have a pharmacological effect).

Also, a drug can be designed using the selected candidate substance as an active ingredient by the method for screening a substance having a high affinity with the target substance of the present invention. In particular, in the design of molecular targeted drugs, it is possible to eliminate the elucidation of the cause of the disease at the molecular level or genome level by simplifying the selection of the compound library, and to simplify the molecular design of the drug. Improvement can be expected.

In addition, a drug can be produced using the selected candidate substance as an active ingredient by the method for screening a substance having a high affinity for the target substance of the present invention.

Claims (15)

Preparing a measurement sample by contacting a sample containing a target substance and a candidate substance;
Performing an imaging mass spectrometry method using the MALDI method with the target substance as a matrix on the measurement sample, and identifying ionized molecules;
Selecting the identified molecule as a substance having a high affinity for the target substance, and screening a substance having a high affinity for the target substance. The step of preparing the measurement sample includes
Contacting a test sample containing the candidate substance with the target substance;
Drying the contacted sample and the target substance in a contact state to obtain a measurement sample, and
The identification step performs a spectrum scanning step of obtaining an ionized molecule spectrum by performing imaging mass spectrometry using MALDI method on the measurement sample,
Identifying a peak substance from the spectrum,
The method of claim 1. The method according to claim 1 or 2, wherein the target substance is a compound having a molecular weight of 10,000 or less. The method according to any one of claims 1 to 3, wherein the candidate substance is a phospholipid. 5. The sample according to claim 1, wherein the sample containing the candidate substance is a tissue section, and the measurement sample is obtained by attaching the target substance to the surface of the sample containing the candidate substance. the method of. The wavelength of the laser used for imaging mass spectrometry using the MALDI method is 200 to 400 nm,
The method according to any one of claims 1 to 5, wherein the target substance is a compound having a molecular weight of 300 to 10,000 containing an aromatic ring and / or a heterocyclic ring. A substance different from the target substance is used as a control substance, and
Preparing a control sample for measurement in which the sample containing the control substance and the candidate substance is contacted;
Performing an imaging mass spectrometry method using the MALDI method with the control substance as a matrix on the measurement control sample, and identifying ionized molecules;
Selecting the identified molecule as a substance having a high affinity with the control substance,
The molecule selected from the molecules ionized from the measurement sample other than the molecule selected as a substance having a high affinity with the control substance is selected as a substance having a high affinity with the target substance. The method according to any one of 1 to 6. The control substance is 3,5-methoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, trans-4-hydroxy-3-methoxycinnamic acid, 2,5-dihydroxybenzoic acid, 8. The method of claim 7, which is 3-hydroxypicolinic acid, 9-aminoacridine, 2,5-dihydroxyacetophenone, or 1,8-dihydroxy-9,10-dihydroanthracen-9-one. Contacting a test sample containing a target substance with a candidate substance;
Drying the test sample and the candidate substance in a contact state to obtain a measurement sample;
A scanning step of performing imaging mass spectrometry using MALDI method on the measurement sample and scanning m / z corresponding to the target substance among the ionized molecules;
A peak intensity measuring step for measuring the peak intensity at the m / z;
Selecting the candidate substance in which the peak intensity is detected as a substance having a high affinity with the target substance.
A method for screening a substance having high affinity with a target substance. The method according to claim 9, wherein the candidate substance is a compound having a molecular weight of 10,000 or less. The method according to claim 9 or 10, wherein the target substance is a phospholipid. The sample according to any one of claims 9 to 11, wherein the sample containing the target substance is a tissue section, and the sample for measurement is obtained by attaching the candidate substance to the surface of the sample containing the target substance. the method of. The wavelength of the laser used for imaging mass spectrometry using the MALDI method is 200 to 400 nm,
The method according to any one of claims 9 to 12, wherein the candidate substance is a compound having a molecular weight of 300 to 10,000 containing an aromatic ring and / or a heterocyclic ring. A screening step for a substance having high affinity with the target substance according to any one of claims 9 to 13,
A method for designing a medicine, comprising containing a candidate substance selected by the screening step as an active ingredient. A screening step for a substance having high affinity with the target substance according to any one of claims 9 to 13,
A method for producing a medicine, comprising containing a candidate substance selected by the screening step as an active ingredient.

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