Comprehensive screening method of compound strongly coupled to target compound

Abstract

To provide a technique for comprehensively screening a compound which can be irreversibly strongly coupled to a target compound containing protein.SOLUTION: A screening method for screening a candidate compound which can be irreversibly coupled to a target compound includes: a step (i) of immobilizing a candidate compound onto a substrate as a protein-compound composite body, through protein for immobilization for immobilizing the compound onto the substrate, in which the protein for immobilization is protein in blood or its analog; a step (ii) of bringing the target compound into contact with the candidate compound immobilized onto the substrate; a step (iii) of cleaning the substrate; and a step (iv) of detecting the target compound coupled to the candidate compound.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for comprehensively screening candidate compounds that strongly bind to target compounds including proteins and the like. [Background technology]

[0002] The binding of proteins and other substances to compounds provides important information for elucidating biological phenomena and biological reactions (especially the effects of drugs on living organisms). For example, when developing a drug that inhibits a specific enzyme or the infection of a specific virus, if there is a compound that strongly and irreversibly binds to the enzyme or protein required for viral infection, it could be a candidate compound for a useful therapeutic drug. Covalent drugs (covalent inhibitors), which have been attracting attention in recent years, are also drugs developed based on this concept, and drugs such as aspirin and penicillin have long been used as representative examples of covalent drugs.

[0003] "Compound microarrays" are known as a method for evaluating the physical interaction between a target protein and a compound. Inspired by DNA microarrays, "compound microarrays" are immobilized on a chip (or glass slide) instead of DNA, and are used to evaluate the physical interaction between a target protein and a compound. Compound microarray technology is superior in principle as a technology for efficiently utilizing a compound library, and various studies have been conducted on immobilizing compounds on glass slides or special substrates (e.g., Non-Patent Document 1).

[0004] The inventors have previously developed a method related to compound microarrays, which allows compounds with a wide variety of structures and functional groups to be easily immobilized onto a substrate while maintaining their structure, without the need for modification (Patent Document 1). However, at present, there are almost no techniques for comprehensively screening compounds that strongly and irreversibly bind to proteins, etc., such as the above-mentioned covalent drugs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-189080 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of the Society of Organic Synthesis Vol.64 No.6 35-46.(2006) Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a technique for comprehensively screening candidate compounds that can irreversibly bind to target compounds including proteins. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors purchased a commercially available protein microarray, HuProt Human Proteome Microarray v3.0 (CDI Labs), and verified the washing method in the steps of the compound screening method according to the present invention. The commercially available protein microarray is equipped with an IgG antibody fluorescently labeled with Alexa Fluor 555 / 647, but by performing the harsh washing conditions according to the present invention (specifically, shaking washing with a shaker (1 minute x 3 times), rinsing washing, shaking washing with a shaking table (3 minutes), and rinsing washing), all of the IgG antibodies mounted on it were washed away. In the conventional compound microarray technology, the washing step after the interaction between compounds needs to be under mild conditions so that the compounds immobilized on the array chip do not dissociate, and under the harsh conditions described above, the compound group containing the protein immobilized on the array chip dissociated (see Comparative Example below). Therefore, when the present inventors used the compound microarray immobilization technology developed by the present inventors, it was surprisingly found that the compounds containing the protein immobilized on the substrate could withstand the harsh washing steps described above. As a result, the present invention has succeeded in developing a compound microarray technology for comprehensively screening candidate compounds that can irreversibly bind to a target compound. The present invention has been completed based on this finding, and includes the following aspects: That is, one aspect of the present invention is [1] A method for screening a candidate compound capable of irreversibly binding to a target compound, comprising the steps of: (i) immobilizing the candidate compound on a substrate as a protein-compound complex via an immobilizing protein for immobilizing the compound on the substrate, the immobilizing protein being a blood protein or an analog thereof; (ii) contacting the target compound with the candidate compound immobilized on the substrate; (iii) cleaning the substrate; (iv) detecting the target compound bound to the candidate compound; The present invention relates to a screening method comprising the steps of: Here, one embodiment of the screening method of the present invention is [2] The screening method according to [1] above, wherein the washing step (iii) is (iii') shaking and cleaning the substrate; (iii'') rinsing the substrate; The present invention is characterized by comprising: Moreover, one embodiment of the screening method of the present invention comprises: [3] The screening method according to [2] above, The method is characterized in that it comprises, after the step (iii''), of rinsing the substrate, a step (iii''') of placing the substrate in a cleaning solution. Moreover, one embodiment of the screening method of the present invention comprises: [4] The screening method according to any one of [1] to [3] above, The method is characterized in that, before the step (iv), the steps (iii') and (iii''), or the steps (iii'), (iii'') and (iii'') are repeated at least two times. Moreover, one embodiment of the screening method of the present invention comprises: [5] The screening method according to any one of [1] to [4] above, the compound of interest is a protein or a portion thereof; The candidate compound is characterized in that it is a low molecular weight compound. Moreover, one embodiment of the screening method of the present invention comprises: [6] The screening method according to any one of [1] to [5] above, the target compound is a protein or a portion thereof derived from a pathogen, The candidate compound is characterized in that it is a compound known as an active ingredient of a therapeutic or prophylactic drug. Moreover, one embodiment of the screening method of the present invention comprises: [7] The screening method according to any one of [1] to [6] above, The blood protein or its analog is characterized in that it is serum albumin, alpha globulin, transferrin, lysozyme, apolipoprotein, transsilentin, ovalbumin, lactalbumin, ovotransferrin, or lactotransferrin. Moreover, one embodiment of the screening method of the present invention comprises: [8] The screening method according to any one of [1] to [7] above, The protein-compound complex is characterized in that the protein for immobilization and the candidate compound are directly bound to each other. Moreover, one embodiment of the screening method of the present invention comprises: [9] The screening method according to any one of [1] to [8] above, The method further comprises a step of subjecting the surface of the substrate to a blocking treatment prior to the step (ii). Moreover, one embodiment of the screening method of the present invention comprises:

[10] The screening method according to any one of [1] to [9] above, The immobilization step comprises: (a) mixing the protein to be immobilized and the compound in a solvent to prepare a protein-compound complex; (b) immobilizing the protein-compound complex on the substrate; The present invention is characterized by comprising: Effect of the Invention

[0009] According to the screening method of the present invention, it is possible to screen for compounds that can bind strongly and irreversibly to a target compound such as a protein. [Brief description of the drawings]

[0010] [Figure 1]The table in Figure 1 shows the top 10 compounds that showed strong binding to the novel coronavirus S protein, which was screened using the screening method of the present invention. The values ​​in the table indicate the fluorescence intensity when the novel coronavirus S protein bound to each compound was detected with a labeled antibody. [Diagram 2] FIG. 2 shows the results of measuring the fluorescence of a control protein (Alexa Fluor 555-labeled IgG) on a commercial protein microarray before washing in Comparative Example 1 below (FIG. 2A) and on a commercial microarray after washing in Comparative Example 1 below (FIG. 2B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. A method for screening a candidate compound capable of irreversibly binding to a target compound 1-1. Overview One aspect of the present invention relates to a method for comprehensively screening candidate compounds capable of strongly and irreversibly binding to a target compound. The screening method of the present invention comprises the following steps: (i) immobilizing the candidate compound on a substrate as a protein-compound complex via an immobilizing protein for immobilizing the compound on the substrate, the immobilizing protein being a blood protein or an analog thereof; (ii) contacting the target compound with the candidate compound immobilized on the substrate; (iii) cleaning the substrate; (iv) detecting the target compound bound to the candidate compound. According to the screening method including the above steps (i) to (iv), it is possible to screen for a candidate compound that can irreversibly and strongly bind to a target compound. The embodiments of each step are described in detail below, but the above steps (i), (ii), and (iv) can be carried out with reference to, for example, JP 2021-189080 A (Patent Document 1).

[0012] 1-2.Definition As used herein, the term "capable of binding irreversibly" includes, but is not limited to, compounds binding to each other through an irreversible reaction (e.g., formation of an irreversible covalent bond), and also includes a state in which the compounds are strongly bound to each other such that the bond between them can be maintained even after the washing step (iii) in the screening method of the present invention. In a preferred embodiment, the screening method of the present invention is a method for screening a candidate compound that forms an irreversible covalent bond with a target compound.

[0013] In the present specification, the term "substrate" refers to a material capable of immobilizing a protein-compound complex via a protein for immobilization, and is not limited as long as it can be used for the detection of a compound immobilized on the substrate. Examples of such substrates include inorganic materials such as metals, metal oxides, glass, quartz, silicon, and ceramics, synthetic polymers such as elastomers, plastics, polyester resins, polyethylene resins, polypropylene resins, ABS resins, nylon, acrylic resins, fluororesins, polycarbonate resins, polyurethane resins, methylpentene resins, phenolic resins, melamine resins, epoxy resins, and vinyl chloride resins, and natural polymers such as chitin, chitosan, and cellulose. The shape of the substrate is not limited, and examples thereof include flat shapes such as flat plates, flat membranes, films, and porous membranes, and three-dimensional shapes such as cylinders, stamps, multiwell plates, microchannels, and fine particles. As the substrate, substrates made of glass, quartz, or silicon are more preferable, and glass is even more preferable. Glass is a transparent compound whose main component is silicate. The glass that can be used for the substrate of the present invention is not limited as long as it can immobilize a compound via an immobilizing protein, and may contain chemical components other than silicate. Examples of the glass include, but are not limited to, soda-lime glass, borosilicate glass, and lime glass. From the viewpoint of industrial use, a glass plate or a slide glass is preferable, but is not limited to the following. As the slide glass, a slide glass coated with poly-L-lysine, aminosilane, or the like, or a slide glass into which amino groups are introduced at a high density can also be used. As the coated slide glass, a commercially available product (for example, manufactured by Matsunami Glass Industry Co., Ltd.) can be used.

[0014] The "protein for immobilization for immobilizing a compound on a substrate" that can be used in the present invention is not limited as long as it is a protein that can immobilize a compound on a substrate (preferably glass) as defined in the present specification, and examples thereof include blood proteins. Such proteins for immobilization are not limited to those that are biosynthesized, and chemically synthesized proteins can also be used. The origin of the protein for immobilization is not limited, and it is preferably a blood protein derived from a mammal (human, monkey, cow, sheep, rabbit, rat, guinea pig, mouse, etc.) or an avian species. In this specification, the term "protein for immobilization" includes modified forms such as mutants and modified forms, or parts thereof, as long as the protein has the property of immobilizing a compound on a substrate. The term "mutant" refers to, for example, a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence constituting the protein for immobilization, and has the property of forming a complex with a compound to immobilize the compound on a substrate. The term "modified form" refers to a protein in which a part of the amino acid sequence of the protein is chemically modified to add a chemical structure other than the amino acid sequence of the protein for immobilization, or a protein in which a part of the chemical structure of the protein for immobilization has been removed, and has the property of forming a complex with a compound to immobilize the compound on a substrate. As used herein, "blood protein" refers to a protein contained in blood. Examples of blood proteins that can be used in the present invention include albumin (serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA)), α-globulins (α1-antitrypsin, haptoglobin, α2-macroglobulin, ceruloplasmin, thyroxine-binding globulin, etc.), transferrins (apo-transferrin, holo-transferrin, etc.), lysozyme, apolipoprotein, transsilentin, etc. In addition, some of these blood proteins are also included as long as they can form a complex with a compound and be immobilized on a substrate. In addition, in this specification, "analog of blood protein" refers to a protein derived from other than blood, which has similar properties to a specific blood protein, or a modified form thereof, or a part thereof, and which can immobilize a compound on a substrate. Examples of the analog of blood protein derived from other than blood include ovalbumin, lactalbumin, ovotransferrin, lactotransferrin, etc. Blood proteins may be isolated from serum collected from a living body, or may be artificially synthesized (including biosynthesis and chemical synthesis). Methods for preparing serum from a living body and isolating and purifying each blood protein from the serum are known. Commercially available blood proteins may also be used. In a more preferred embodiment, the blood proteins used in the present invention are proteins isolated from serum.

[0015] In this specification, the term "compound" includes low molecular weight compounds, high molecular weight compounds, organic compounds, inorganic compounds, polysaccharides, polymers, lipids, nucleic acids and their analogs, polypeptides, proteins, antibodies, lectins, and compounds formed by combinations thereof. In the present invention, the compound immobilized on the substrate is not limited as long as it can be dissolved in a solvent and form a complex with an immobilization protein for immobilizing the compound on the substrate. The compound may be naturally occurring or artificially synthesized. Furthermore, the compounds forming the protein-compound complex may be the same as each other in the protein-compound complex, or may be two or more different compounds.

[0016] "Low molecular weight compound" means a compound with a molecular weight of approximately 10,000 or less, and is distinguished from polymeric compounds, nucleic acids, and polypeptides. Examples include, but are not limited to, amoxicillin, ampicillin, apramycin, bacitracin, cefalexin, ceftazidime, cloxacillin, gentamicin, hygromycin B, neomycin, penicillin G, streptomycin, sulfadimidine, tetracycline, etc. The term "polymeric compound" refers to a compound having a molecular weight of more than about 10,000 and is distinguished from low molecular weight compounds, nucleic acids, and polypeptides. Examples of the polymeric compound include, but are not limited to, starch, cellulose, synthetic fibers, plastics, rubber, asbestos, and phosphonitrile chloride polymers. The term "polysaccharide" refers to a compound in which multiple monosaccharides such as glucose and mannose are polymerized, and is not limited by the type of monosaccharides constituting the polysaccharide, the bonding method, the molecular weight, the form of the main chain and side chain, etc. Examples of polysaccharides include, but are not limited to, acidic polysaccharides such as carrageenan, pectin, gum arabic, xanthan gum, gellan gum, agar, tragacanth gum, etc.; neutral polysaccharides such as tamarind seed gum, guar gum, locust bean gum, starch, pullulan, etc.; and basic polysaccharides such as chitosan, etc. Additionally, "organic compound" refers to a compound in which carbon is the center of atomic bonds, and "inorganic compound" refers to a compound in which carbon is not involved in atomic bonds. "Polymer" refers to a compound formed by polymerization of two or more monomers. "Lipid" refers to an organic compound present in living organisms that is insoluble in water but soluble in organic solvents. Lipids include, but are not limited to, biologically active substances such as prostaglandins. "Nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), etc. The term "polypeptide" refers to a polymer formed by forming peptide bonds between a large number of amino acids through a degenerate reaction. The compounds listed above are not particularly limited and can be the target compounds to be immobilized on a substrate in the screening method of the present invention.

[0017] As used herein, the term "target compound" refers to, but is not limited to, a protein derived from a pathogen or a part thereof (e.g., a part of a domain) or a compound identified as one of the causes of a disease (such as a target protein that can contribute to the treatment of a specific disease). A pathogen refers to a virus, bacterium, fungus, protozoa, phage, etc. that parasitizes a living organism and causes an infectious disease. A protein derived from a pathogen can also be a recombinant protein produced by a microorganism or a lysate (dissolution product) of a pathogen. As used herein, the term "candidate compound" refers to, but is not limited to, a compound known as an active ingredient of a medical drug. In a preferred embodiment, the "candidate compound" may be an active ingredient of a therapeutic drug administered to treat or prevent a specific disease. When a new therapeutic use of such a compound is found by the screening method of the present invention, the safety of administration to the human body is guaranteed, and therefore the compound can be quickly repurposed as a therapeutic drug for the new use. In the screening method of the present invention, by using a compound microarray loaded with existing compounds as candidate compounds, it becomes possible to rapidly screen for compounds that bind strongly and irreversibly to proteins required for pathogenic microorganisms or viruses that cause emerging infectious diseases such as the new coronavirus to infect humans, or compounds that bind strongly to target proteins of diseases for which no effective therapeutic drugs exist. If the compound obtained by the screening method of the present invention is used as an active ingredient in an existing drug, its safety is guaranteed, and therefore it can be rapidly repurposed as a therapeutic drug for the emerging infectious disease or disease.

[0018] 1-3. (i) A step of immobilizing a candidate compound on a substrate as a protein-compound complex via an immobilizing protein for immobilizing the compound on the substrate. Immobilizing a candidate compound on a substrate via an immobilizing protein means that the immobilizing protein binds to the substrate and the candidate compound binds to the immobilizing protein, resulting in immobilization of the candidate compound on the substrate. The method of immobilizing a compound on a substrate in this manner may involve first preparing a complex between the compound and the immobilizing protein and immobilizing the complex on the substrate, or first binding the immobilizing protein to the substrate and then binding the compound to the immobilizing protein. That is, in one embodiment, the immobilization step (i) comprises: (a) mixing a protein to be immobilized and a compound in a solvent to prepare a protein-compound complex; (b) immobilizing the protein-compound complex on a substrate; Includes. In another embodiment, the immobilization step (i) comprises: (a') immobilizing a protein to be immobilized on a substrate; (b') contacting a compound with the protein for immobilization immobilized on the substrate to form a protein-compound complex; Includes. In this specification, the terms "candidate compound" and "target compound" are used for the sake of convenience to distinguish between a compound that is immobilized on a substrate as a protein-compound complex and a compound that is contacted with the compound to determine whether or not it binds to the compound. Combinations of candidate compounds and target compounds are not limited to the following, but for example, the "candidate compounds" can be a group of compounds known as therapeutic or preventive drugs, and the "target compounds" can be proteins required when pathogenic microorganisms or viruses infect human cells. On the other hand, the "candidate compounds" can be a group of proteins required when pathogenic microorganisms or viruses infect human cells, and the "target compounds" can be a group of compounds known as therapeutic or preventive drugs.

[0019] In one embodiment, the process of immobilizing a candidate compound on a substrate of the present invention via an immobilizing protein includes the above-mentioned step (a) of mixing the immobilizing protein and the compound in a solvent to prepare a protein-compound complex. The solvent that can be used in step (a) is not limited as long as the protein to be immobilized and the compound can be dissolved and a protein-compound complex can be formed in the solvent. Suitable examples of the solvent include those that are commonly used for immobilization or detection in detection techniques for various compounds immobilized on a substrate, such as compound microarrays, DNA microarrays, and protein microarrays. Such solvents are well known and commercially available ones may be used. Examples of the solvent include, but are not limited to, distilled water, dimethyl sulfoxide (DMSO), methyl acetate, acetone, or mixtures thereof. The conditions for mixing the protein for immobilization and the compound in the solvent are not limited as long as a protein-compound complex can be formed. For example, the protein and the compound may be added to the same solvent and mixed by standing or shaking for about 10 minutes to 1 hour, but are not limited thereto. The conditions for mixing vary depending on the protein for immobilization and the compound used, and can be appropriately set by those skilled in the art. The concentrations of the protein for immobilization and the compound are also not limited as long as a protein-compound complex can be formed, and can be appropriately set for each protein for immobilization used and each compound of interest. The concentration of the protein for immobilization can be within the range of 0.1 mg / ml to 10 mg / ml, but is preferably within the range of 0.4 mg / ml to 1.0 mg / ml. The solvent may further contain additives such as stabilizers.

[0020] As used herein, the term "protein-compound complex" refers to a complex formed by binding a compound to a protein for immobilization for immobilizing a compound on a substrate in a solvent. Thus, in a protein-compound complex, the protein for immobilization and the compound are directly bound to each other without a spacer or the like therebetween. The protein for immobilization and the compound may be bound in a one-to-one ratio, or in a one-to-multiple, multiple-to-one, or multiple-to-multiple ratio. In one embodiment, a "protein-compound complex" refers to a complex formed by binding between a protein and a compound without a specific binding mode between a specific atom or a specific structure. In other words, when there are multiple protein-compound complexes, the immobilization protein and the compound are bound between different atoms or structures in each protein-compound complex. As a result, the compound forming the complex binds to the immobilization protein in a different orientation between the protein-compound complexes and presents its surface to the outside in a different orientation between the protein-compound complexes. In one embodiment, the "protein-compound complex" can be a complex in which the immobilization protein and the compound are bound by a non-covalent bond. In a preferred embodiment, the "protein-compound complex" refers to a complex in which the immobilization protein and the compound are bound by physical adsorption.

[0021] In one embodiment, the step of immobilizing a compound on a substrate of the present invention via an immobilizing protein includes, after the above step (a), a step (b) of immobilizing the protein-compound complex on a substrate. As used herein, "immobilizing a protein-compound complex on a substrate" refers to immobilizing the complex on a substrate via the structure of the immobilizing protein in the protein-compound complex. The immobilization on the substrate may be performed with a strength sufficient for the intended use, and may be performed with a strength sufficient to enable detection of a candidate compound that can irreversibly bind to the target compound. In one embodiment, the protein-compound complex may be bound to the substrate via the structure of the immobilizing protein by a non-covalent bond. In a preferred embodiment, the protein-compound complex is bound to the substrate via the structure of the immobilizing protein by physical adsorption. The protein-compound complex can be immobilized by contacting a solution containing the protein-compound complex with a substrate. The solution containing the protein-compound complex may be the solution prepared in the above step (a) as it is, or may be transferred to another solvent before use. The method of contacting a solution containing a protein-compound complex with a substrate is not limited as long as the solution can be contacted with the substrate and the protein of the protein-compound complex can be immobilized on the substrate. Preferably, the solution is spotted on the substrate using a known ultra-microdispensing device (MicroDiagnostic) or the like.

[0022] The compound in the protein-compound complex immobilized on the substrate by the immobilization step (b) maintains its structure. The compound maintains its structure means that the compound does not have any modification for immobilization in its structure. The compound in the protein-compound complex maintains all or part of its structure in the same manner as before the protein-compound complex is formed. For example, by detecting the compound after the protein-compound complex is formed using an appropriate detection means, it can be confirmed that the compound maintains the structure required for detection. An example of the compound in the protein-compound complex maintaining its structure includes, but is not limited to, the structure of the epitope to which an antibody against the compound binds. In a preferred embodiment, the compound in the protein-compound complex immobilized on the substrate maintains its activity. The compound maintaining its activity includes maintaining the activity required for detecting the compound in the same manner as before the protein-compound complex was formed. The activity is preferably the same as before the protein-compound complex was formed, but it is sufficient that the activity is maintained to the extent required for detecting the compound. As a non-limiting example, when the compound is an enzyme, the activity of the enzyme is maintained, and in this case, the compound (enzyme) immobilized on the substrate can be detected by utilizing the enzyme activity.

[0023] After contacting the substrate with a solution containing a protein-compound complex, it is preferable to wash the substrate as necessary to remove impurities that have not been immobilized on the substrate. Therefore, in one embodiment, the method for immobilizing a compound on a substrate of the present invention can include a washing step after step (b). This can improve the sensitivity and accuracy of detection of the compound immobilized on the substrate. The solution used for washing the substrate is not limited as long as it can remove impurities without completely dissociating the bond between the protein-compound complex and the substrate, and any known washing solution that can be used in the manufacture of microarray chips, etc. Specific examples include, but are not limited to, ethanol, ultrapure water, etc. The washing step is preferably performed by immersing the substrate on which the protein-compound complex is immobilized in a washing solution such as ethanol or ultrapure water and shaking it for about 30 seconds at room temperature, although this is not limited thereto. The washing step is preferably performed once or multiple times, and for example, washing with ethanol can be performed once, followed by washing with ultrapure water twice.

[0024] In one embodiment, the step of immobilizing a compound on a substrate of the present invention via an immobilizing protein includes the step of (a') immobilizing the immobilizing protein on a substrate. The protein for immobilization can be immobilized on the substrate by contacting a solution containing the protein for immobilization on the substrate. The method of contacting the solution containing the protein for immobilization with the substrate is not limited as long as the protein for immobilization can be immobilized on the substrate by contacting the solution with the substrate. A preferred form is spotting on the substrate using a known ultra-microdispensing device (MicroDiagnostic) or the like. The solvent can be any solvent that can be used in the above step (a).

[0025] In one embodiment, the process of immobilizing a compound on a substrate of the present invention via an immobilization protein includes, after the above-mentioned process (a'), a process (b') of contacting a compound with the immobilization protein immobilized on the substrate to form a protein-compound complex. The method of contacting the protein for immobilization immobilized on the substrate with the compound may be to contact the protein for immobilization on the substrate with a solution containing the compound. Preferably, the compound is spotted on the substrate using a known ultra-microdispensing device (MicroDiagnostic) or the like. The conditions for contacting the solution containing the compound with the protein for immobilization on the substrate can be appropriately set using the same solvent and mixing conditions as in the method of step (a) above. In one embodiment, the method for immobilizing a compound on a substrate of the present invention may include a washing step after the step (a') and / or (b'). The washing step may be carried out under the same conditions as the washing step after the step (b).

[0026] 1-4.(ii) A step of contacting a target compound with a candidate compound immobilized on a substrate Methods for contacting a target compound with a candidate compound immobilized on a substrate as a protein-compound complex are known, and the conditions such as the solvent, temperature, and time used during contact can be set appropriately depending on the combination of the candidate compound and the target compound that constitute the protein-compound complex. As the solvent, for example, distilled water, dimethyl sulfoxide (DMSO), methyl acetate, acetone, or a mixture thereof can be used. In addition, a commercially available solution for cleaning compound microarrays (for example, solution A (Fukushima Protein Factory, Fukushima, Japan), a dedicated solution for compound microarrays) can also be used. For example, the target compound can be added to a commercially available solution A (Fukushima Protein Factory, Fukushima, Japan) dedicated to compound microarrays, and the substrate can be immersed in the solution containing the target compound to bring the candidate compound into contact with the target compound. When immersing the substrate in the solution, a known method for promoting contact between compounds, such as shaking, may be appropriately adopted. The compounds can be brought into contact with each other for 1 to 30 hours under temperature conditions of, but not limited to, room temperature to 40°C. For example, when using a protein of about 150 kDa (such as S protein derived from the new coronavirus), the protein in the solution can be at a concentration of 1 ng / mL to 1 μg / mL.

[0027] Before contacting the target compound, blocking treatment may be performed to suppress non-specific binding or to increase the stability of the immunoassay reagent itself. The blocking treatment can be performed according to a known method using a known or commercially available blocking agent used in immunoassay methods such as ELISA. The concentration of the blocking agent can be appropriately determined, but is preferably 0.1% to 5%, more preferably 1% to 3%. The treatment temperature can also be appropriately determined, but is preferably 4°C to 40°C, more preferably 20°C to 30°C. The treatment time can also be appropriately determined, but is preferably 0.5 hours to 24 hours, more preferably 0.5 hours to 3 hours. For example, blocking treatment can be performed by immersing the substrate in commercially available Blocking One (Nacalai tesque, Kyoto, Japan) and leaving it to stand or shake it for about 30 minutes to 12 hours under conditions of 4°C to 40°C. After the blocking reaction, it is preferable to wash the substrate. In the washing step, a solvent that can be used in the above step (a) (e.g., distilled water, dimethyl sulfoxide (DMSO), methyl acetate, acetone, or a mixture thereof) can be used.

[0028] 1-5.(iii) Step of cleaning the substrate After contacting the target compound with the candidate compound immobilized on the substrate, the substrate can be washed with a washing solution to remove from the substrate target compounds that are not bound to the candidate compound, target compounds that are weakly bound to the candidate compound, or target compounds that are reversibly bound to the candidate compound. Examples of the cleaning solution include distilled water, dimethyl sulfoxide (DMSO), methyl acetate, acetone, and mixtures thereof. Commercially available cleaning solutions for chemical microarrays (e.g., solution A (Fukushima Protein Factory, Fukushima, Japan)), which is a dedicated solution for chemical microarrays, can also be used. Washing the substrate with a washing solution includes a step of shaking and washing the substrate (a shaking and washing step) and a step of rinsing the substrate with the washing solution after shaking and washing (a rinsing step). By repeating the shaking and washing step and the rinsing step, it is possible to screen a candidate compound that can strongly and irreversibly bind to the target compound.

[0029] The shaking washing step is performed by repeating shaking washing several times using a commercially available shaker or shaking table. The shaker or shaking table is preferably one that generates horizontal eccentric vibration. The conditions of shaking washing are not limited as long as the target compound other than the target compound irreversibly bound to the candidate compound can be removed from the substrate. For example, the shaking speed is preferably 20 to 300 r / min, more preferably 50 to 150 r / min. The shaking width is preferably 10 to 50 mm, more preferably 20 to 30 mm, but is not limited thereto. The temperature of the washing solution during shaking washing is preferably room temperature to 40°C, more preferably 20 to 27°C. The shaking washing time is preferably 1 minute or more (e.g., 1 to 30 minutes, 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, 1 to 5 minutes, 1 to 3 minutes), and the shaking washing is preferably repeated two or more times (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more times).

[0030] After the shaking washing, it is preferable to include a step of washing and removing the unbound target compound, the target compound weakly bound to the candidate compound, or the target compound reversibly bound to the candidate compound from the substrate using a washing solution (rinsing step). The method of washing away the unbound target compound is not limited, and examples thereof include a method of putting the substrate in and out of a new washing solution, and a method of pouring a new washing solution onto the substrate. In this case, the temperature of the washing solution is preferably room temperature to 40°C, and more preferably 20 to 27°C. It is preferable to repeat the method of washing away the unbound target compound, the target compound weakly bound to the candidate compound, or the target compound reversibly bound to the candidate compound with the washing solution two or more times (for example, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times). After washing away the unbound target compound with the washing solution, it is preferable to leave the substrate standing in the washing solution for a while. That is, the screening method of the present invention includes a step of leaving the substrate standing in the washing solution after the step of rinsing the substrate. The time for leaving the substrate standing is not limited to the following, and can be 1 to 30 minutes, 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, or 1 to 5 minutes.

[0031] The shaking washing step and the rinsing step may be repeated multiple times (e.g., two or more times, three or more times, four or more times, five or more times) as one set. When the shaking washing step and the rinsing step are repeated as one set, the conditions of each washing step from the second set onwards may be the same as or different from those of the first set. In a preferred embodiment, the shaking washing step and the rinsing step are repeated twice. In one embodiment of the step of removing target compounds other than the target compound strongly bound to the candidate compound from the substrate, the shaking washing step and the rinsing step are repeated at least twice. Another embodiment includes the shaking washing step, the rinsing step, the shaking washing step, and the rinsing step in this order.

[0032] 1-6. (iv) Detecting the target compound bound to the candidate compound In this step, the target compound that is still bound to the candidate compound immobilized on the substrate as a protein-compound complex after passing through the washing step (iii) above is detected. The detection method of the target compound can be appropriately adopted using a known detection means that is suitable for the target compound. Such detection means are not limited as long as they are capable of detecting the presence of the compound by binding or reacting with the compound. Examples of such detection means include, but are not limited to, antibodies, lectins, probes, receptors, antigens, haptens, nucleic acids (DNA, RNA, or PNA), sugar chains, ligands, aptamers, polynucleotides, lipids, enzymes, enzyme substrates, and the like. In addition, known methods can be adopted as a method for detecting the compound immobilized on the substrate using these detection means. Although not limited to the following methods, a detection method using a labeled antibody can be used as a detection method in a preferred embodiment.

[0033] 1-7. Recovery process In one embodiment, the step of immobilizing a compound on a substrate of the present invention via an immobilizing protein includes a step of recovering the compound from the substrate after the detection step. The method of recovering the compound from the substrate may involve recovering the compound as a protein-compound complex by separating the protein-compound complex from the substrate, or recovering the compound itself by separating it from the protein-compound complex. The method of recovering the compound may employ any known means appropriate for the substrate and protein used, and the type of compound to be recovered.

[0034] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0035] (Example 1. Screening of compounds capable of irreversibly binding to the novel coronavirus S protein) 1-1. Fixation of compounds 1,600 types of compounds used as active ingredients in existing drugs were mixed with human serum albumin (HSA) (Sigma-Aldrich) and printed on a glass slide. More specifically, each compound was dissolved in solution A (Fukushima Protein Factory) for protein microarrays to a concentration of 0.5 mM. HSA was also prepared to a final concentration of 0.1 micrograms / microliter and mixed with solution A containing the compound to form a compound-HSA complex. Solution A containing the compound-HSA complex was spotted on a glass slide as microdroplets of approximately 2 to 4 nl using an ultra-microdispensing device (MicroDiagnostic), and the compound-HSA complex was printed on the glass slide substrate.

[0036] The slide glass on which the compounds were printed (hereinafter referred to as "compound microarray") was immersed in ethanol and shaken at room temperature for 30 seconds. The compound microarray was then immersed in ultrapure water and shaken at 26°C for 30 seconds. Shaking in ultrapure water was performed twice. To replace the solution, the compound microarray was immersed in solution A (Fukushima Protein Factory, Fukushima, Japan) for protein microarrays and shaken at 26°C for approximately 5 minutes.

[0037] 1-2. Addition of novel coronavirus S protein The compound microarray treated as described above was immersed in Blocking One (Nacalai tesque, Kyoto, Japan) and shaken at 26° C. for 1 hour. Next, solution A containing the novel coronavirus S protein was prepared at a concentration of 50 ng / ml, and 100 ng of the novel coronavirus S protein was applied to the compound microarray. After applying the novel coronavirus S protein, the array was shaken at 37°C for 17 hours. The seven types of novel coronavirus S protein used in this test were as shown in the table below.

[0038] [Table 1]

[0039] 1-3.Removal of S protein After contacting the compound microarray with the novel coronavirus-derived S protein, the compound microarray was placed in a polystyrene case (Sanplatec 02271C polystyrene square case No. 11) containing 10 ml of solution A, and was shaken and washed (individually washed) for 1 minute at 26 ° C and 90 r / min in a medium-sized thermostatic shaking incubator Bioshaker (trademark registered) BR-43F (TAITEC). After shaking and washing for 1 minute, the compound microarray was transferred to a new polystyrene case containing 10 ml of solution A, and was shaken and washed again for 1 minute at 26 ° C and 90 r / min in a shaker. Individual washing with a polystyrene case was performed a total of three times. Next, the compound microarray was placed in a metal cage and placed in a staining tray (Kartell 353 staining tray (PMP)) containing 280 ml of solution A. The metal cage was moved up and down 10 times, and then left to stand in solution A for 5 minutes. After that, it was placed on a shaking table (Sakura Seiki Co., Ltd. VF-5 Sakura Vibrator) and shaken for 3 minutes (bulk washing). After 3 minutes, the metal basket was transferred to a new staining vat containing 280 ml of solution A. The metal basket was again moved up and down 10 times, placed on the shaking table, and shaken for washing. Bulk washing using the metal basket was performed a total of 3 times.

[0040] 1-4. Detection using labeled antibodies Anti-6X His tag (registered trademark) (ab9108, GR91766-6; abcam) was diluted to 1 microgram / milliliter in a primary antibody diluent for protein microarrays (Fukushima Protein Factory).

[0041] Two milliliters of the diluted primary antibody solution was added to a protein microarray cassette P-DE1 (Fukushima Protein Factory). The compound microarray was immersed in the solution, the cassette was closed with a lid, and the cassette was sealed with a special clip and reacted at 37°C for about 1 hour with shaking.

[0042] After reacting with the primary antibody, the compound microarray was immersed in solution A for protein microarrays and shaken for 1 minute at 26° C. This process was repeated three times.

[0043] A 1 / 500th amount of Alexa Fluor647-labeled anti-Mouse IgG antibody (711-605-152; Jackson) was added to a secondary antibody diluent (Fukushima Protein Factory) for protein microarrays to give a concentration of 6 micrograms / milliliter, and 2 milliliters of the solution was added to a protein microarray cassette P-DE1. The compound microarray was immersed in the solution, the cassette was closed with a lid, and the cassette was sealed with a special clip, and the reaction was allowed to proceed at 26°C for about 1 hour while shaking.

[0044] After reacting with the secondary antibody, the compound microarray was immersed in solution A for protein microarrays and shaken for 1 minute at 26° C. This process was repeated three times.

[0045] The compound microarray was immersed in solution B (Fukushima Protein Factory) for protein microarrays and shaken for 3 minutes at 26° C. This process was repeated three times.

[0046] The compound microarray was immersed in a final washing solution for protein microarrays (Fukushima Protein Factory) and shaken for 3 minutes at 26° C. This process was repeated four times. 1-5. Negative control As negative controls, the following two tests were performed: (1) A test group in which the reaction was carried out in the same manner as above, except that solution A, which does not contain the new coronavirus S protein, was used in the above "1-2. Addition of the new coronavirus S protein"; and (2) Test group in which the reaction was carried out in the same manner as above, except that in the above "1-2. Addition of novel coronavirus S protein", solution A not containing novel coronavirus S protein was used, and in the above "1-4. Detection using labeled antibody", a primary antibody dilution solution not containing Anti-6X His tag was used.

[0047] 1-6.Measurement of fluorescence intensity After drying the compound microarray, it was centrifuged at 1,500 rpm for 1 minute, and then the fluorescence intensity was scanned using a GenePix 4000B (Molecular Devices, CA, USA). Figure 1 shows the results of extracting 10 compounds with high average fluorescence intensity when reacted with each novel coronavirus S protein. The results in Figure 1 exclude compounds that showed a fluorescence intensity value of 100 or more in the negative control. The red intensity in Figure 1 indicates the fluorescence intensity derived from the Alexa Fluor 647-labeled anti-Mouse IgG antibody. In Figure 1, the "average" indicates the average fluorescence intensity for the seven novel coronavirus S proteins.

[0048] As shown in Figure 1, we were able to screen Noscapine HCl as a compound that may bind strongly and irreversibly to the S protein derived from the new coronavirus. Noscapine HCl is already used as an active ingredient in cold medicines sold in Japan, and is known to be effective against colds, bronchial asthma, acute bronchitis, chronic bronchitis, pneumonia, etc. In other words, since safety for administration to the human body is guaranteed, it can be quickly repurposed as a therapeutic drug. In particular, it has been reported that when the new coronavirus infects alveolar epithelial cells, detachment and necrosis of the alveolar epithelial cells occur, impairing the oxygenation function and causing severe injury. Therefore, it is expected that the compound in question may be able to neutralize (neutralize) the new coronavirus by binding to it in the bronchi and lungs.

[0049] (Comparative Example 1. Examination of the washing process using a commercially available microarray) In this comparative example, the cleaning method according to the present invention was examined using a commercially available proteome microarray (HuProt Human Proteome Microarray v3.0, manufactured by CDI Laboratories). The HuProt Human Proteome Microarray v3.0 is a proteome microarray that contains more than 21,000 proteins and covers ~81% of the human proteome. The microarray is preloaded with control proteins such as Alexa Fluor 555 / 647-labeled IgG. The microarray is used to comprehensively verify the interactions between proteins on the array and objects of interest such as antibodies, serum antibodies, and fluorescently labeled nucleic acids and proteins.

[0050] In order to verify that the washing step according to the present invention is performed under stricter conditions than usual, it was verified whether the commercially available proteome microarray could withstand the washing step in Example 1. Specifically, the commercially available proteome microarray was washed under the same conditions as the washing step in "1-3. Removal of S protein" above. The commercial microarray that had undergone the washing process was scanned for fluorescence intensity from the Alexa Fluor 647-labeled anti-Mouse IgG antibody using GenePix 4000B (Molecular Devices, CA, USA), but no fluorescence was detected. Figure 2 shows the results of observing the fluorescence of a control protein (Alexa Fluor 555-labeled IgG) on the microarray before washing (Figure 2A) and on the microarray after washing (Figure 2B). The results in Figure 2 show that the protein group mounted on the microarray base was dissociated from the microarray base after the washing process of Example 1. Thus, conventional microarrays could not withstand the screening method of the present invention.

Claims

1. A method for screening a candidate compound capable of irreversibly binding to a target compound, comprising the steps of: (i) immobilizing the candidate compound on a substrate as a protein-compound complex via an immobilizing protein for immobilizing the compound on the substrate, the immobilizing protein being a blood protein or an analog thereof; (ii) contacting the target compound with the candidate compound immobilized on the substrate; (iii) cleaning the substrate; (iv) detecting the target compound bound to the candidate compound; A screening method comprising:

2. The screening method according to claim 1 , wherein the washing step (iii) comprises: (iii') shaking and cleaning the substrate; (iii'') rinsing the substrate; A screening method comprising:

3. 3. The screening method according to claim 2, further comprising the steps of: (iii'') rinsing the substrate; (iii''') placing the substrate in a cleaning solution A screening method comprising:

4. The screening method according to claim 2, A screening method, comprising repeating the steps (iii') and (iii'') at least two or more times before the step (iv).

5. The screening method according to claim 1, the compound of interest is a protein or a portion thereof; A screening method, wherein the candidate compound is a low molecular weight compound.

6. The screening method according to claim 1, the target compound is a protein or a portion thereof derived from a pathogen, A screening method, wherein the candidate compound is a compound known as an active ingredient of a therapeutic or prophylactic drug.

7. The screening method according to claim 1, The method of screening, wherein the blood protein or analog thereof is serum albumin, alpha globulin, transferrin, lysozyme, apolipoprotein, transsilentin, ovalbumin, lactalbumin, ovotransferrin, or lactotransferrin.

8. The screening method according to claim 1, The protein-compound complex is a complex in which the protein for immobilization and the candidate compound are directly bound to each other.

9. The screening method according to claim 1, The screening method further comprises a step of performing a blocking treatment on the surface of the substrate before the step (ii).

10. The screening method according to claim 1, The immobilization step comprises: (a) mixing the protein to be immobilized and the compound in a solvent to prepare a protein-compound complex; (b) immobilizing the protein-compound complex on the substrate; A screening method comprising:

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