Method for evaluating activity / reactivity of active / reactive site of protein

Abstract

Provided is a method for evaluating the activity / reactivity of an active / reactive site of a protein, the evaluation method comprising: a labeling step for labeling a protein with an activity-based labeling molecule; and a detection step for detecting, in a solution containing a single molecule of the labeled protein, the activity-based labeling molecule with which the protein is labeled, using an enzymatic reaction of a reporter enzyme that modifies the activity-based labeling molecule. The activity-based labeling molecule is a molecule that reacts with an active / reactive site of the protein and forms a covalent bond with the protein or the active / reactive site thereof after the reaction. The labeling step includes bringing the activity-based labeling molecule into contact with the protein.

Description

Method for evaluating the activity / reactivity of an active / reactive site of a protein

[0001] The present disclosure relates to methods for assessing the activity / reactivity of an active / reactive site of a protein.

[0002] Techniques for detecting the presence of proteins at the single-molecule level are known. For example, digital ELISA is a protein detection method that involves performing a reporter enzyme reaction in an ELISA in a minute volume of solution that can contain a single molecule of protein, and binarizing the results as "signal present" or "no signal" and obtaining the results for a large number of solutions. This method can detect proteins with higher sensitivity than conventional ELISA (Non-Patent Documents 1 and 2). For example, Non-Patent Document 3 discloses a technique for measuring enzyme activity at the single-molecule level.

[0003] U.S. Patent No. 9,797,837

[0004] David M Rissin et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology 28, 595-599 (2010).Soo Hyeon Kim et al., Large-scale femtoliter droplet array for digital counting of single biomolecules, Lab Chip, 2012, 12, 4986.Shingo Sakamoto et al., Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples. Sci. Adv.6,eaay0888(2020).Liu, Y., Patricelli, M.P., and Cravatt, B.F. Activity-based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14695-14699 (1999).Greenbaum, D., Medzihradszky, K.F., Burlingame, A., and Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569-581 (2000).Speers, A.E., and Cravatt, B.F., Activity-Based Protein Profiling (ABPP) and Click Chemistry (CC)-ABPP by MudPIT Mass Spectrometry. Curr. Protoc. Chem. Biol. 1, 29-41. 10.1002 / 9780470559277.ch090138 (2009).Nomura, D.K., Dix, M.M., and Cravatt, B.F., Activity-based protein profiling for biochemical pathway discovery in cancer. Nat. Rev. Cancer 10 (2010), 630-638. 10.1038 / nrc2901.Weerapana, E. et al., Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468 (2010), 790-797. 10.1038 / nature09472.Bar-Peled, L. et al., Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer. Cell 171 (2017), 696-709.e23. 10.1016 / j.cell.2017.08.051.Vinogradova, E. V. et al., An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells. Cell 182 (2020), 1009-1026.e29. 10.1016 / j.cell.2020.07.001.Backus, K.M. et al., Proteome-wide covalent ligand discovery in native biological systems. Nature 534 (2016), 570-574. 10.1038 / nature18002.Canon, J. et al., The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575 (2019), 217-223. 10.1038 / s41586-019-1694-1.Abbasov, M.E. et al., A proteome-wide atlas of lysine-reactive chemistry. Nat. Chem. 13 (2021), 1081-1092. 10.1038 / s41557-021-00765-4. Sun, F., Suttapitugsakul, S., and Wu, R., An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome. Anal. Chem. 93 (2021), 10334-10342. 10.1021 / acs.analchem.1c01935. Yannick Rondelez et al., "Microfabricated arrays of femtoliter chambers allow single molecule enzymology", Nat. Biotech. 23(3), 361-365 (2005).

[0005] While digital ELISA can detect the presence of a protein, it cannot evaluate its activity or reactivity. Furthermore, the method described in Non-Patent Document 3 requires the design and development of an activity detection probe for each enzyme whose activity is to be detected, the probe's signal intensity increasing upon being metabolized by the enzyme. The present disclosure aims to provide a method for evaluating the activity / reactivity of an active / reactive site of a protein.

[0006] The present inventors have devised and conducted extensive research into the application of activity-based protein profiling (ABPP) to the evaluation of proteins at the single molecule level, and as a result have developed a method for evaluating the activity / reactivity of active / reactive sites in proteins by covalently labeling the proteins with activity-based labeling molecules and then detecting the activity-based labeling molecules that have labeled the proteins using an enzymatic reaction of a reporter enzyme that modifies the activity-based labeling molecules.

[0007] One aspect of the present disclosure is, for example, the following [1]: [1] A method for evaluating the activity / reactivity of an active / reactive site of a protein, comprising: a labeling step of labeling a protein with an activity-based labeling molecule; and a detection step of detecting, in a solution containing one molecule of the labeled protein, the activity-based labeling molecule that has labeled the protein using an enzymatic reaction of a first reporter enzyme that modifies the activity-based labeling molecule, wherein the activity-based labeling molecule reacts with the active / reactive site of the protein and, after the reaction, forms a covalent bond with the protein or the active / reactive site thereof, and the labeling step comprises contacting the activity-based labeling molecule with the protein.

[0008] According to the evaluation method [1], like ELISA methods such as digital ELISA, an enzymatic reaction of a first reporter enzyme is used, and the activity / reactivity of a protein's active / reactive site can be evaluated at the single-molecule level using a signal derived from a reaction product generated by the first reporter enzyme metabolizing a substrate as an indicator. According to the evaluation method [1], even if the activity-based labeling molecule itself is not a molecule capable of producing a detectable signal, the enzymatic reaction of the first reporter enzyme can be used to detect that a protein has been labeled with the activity-based labeling molecule. This allows the activity / reactivity of a protein's active / reactive site to be evaluated at the single-molecule level. This allows the activity / reactivity of a protein's active / reactive site to be evaluated at the single-molecule level without the need to newly develop a dedicated activity-based labeling molecule (e.g., a molecule whose signal increases upon reaction with a protein) with a detectable signal for each target protein. Furthermore, since it is possible to simultaneously select an activity-based labeling molecule that is preferable in terms of reactivity with the protein (e.g., reaction rate, selectivity), and a first reporter enzyme that is preferable in terms of signal strength, the activity / reactivity of a protein's active / reactive site can be favorably evaluated at the single-molecule level.

[0009] One aspect of the present disclosure is, for example, the following [2]: [2] The evaluation method according to [1], wherein the labeling step further comprises contacting the protein with a post-translational modification labeling molecule, and the detection step comprises detecting the post-translational modification labeling molecule that has labeled the protein in a solution containing one molecule of the labeled protein, using an enzymatic reaction of a second reporter enzyme that modifies the post-translational modification labeling molecule, and the post-translational modification labeling molecule is a molecule that selectively binds to a post-translational modification site of the protein.

[0010] The evaluation method [2], like ELISA methods such as digital ELISA, uses an enzymatic reaction of a second reporter enzyme and uses a signal derived from a reaction product generated by metabolizing a substrate by the second reporter enzyme as an indicator, thereby simultaneously evaluating the presence or absence of post-translational modification, even in a single-molecule system, thereby enabling evaluation of the effect of post-translational modification on the activity / reactivity of an active / reactive site in a protein.

[0011] One aspect of the present disclosure is, for example, the following [3]: [3] The evaluation method according to [2], wherein the post-translationally modified labeled molecule is a molecule having a molecular weight (g / Mol) of 5,000 or less.

[0012] According to the evaluation method [3], the volume occupied by the post-translational modification labeling molecule in the space surrounding the protein is reduced compared to when an antibody is used as the interaction site for the post-translational modification labeling molecule. This reduces the inhibition of the reaction between the protein and the activity-based labeling molecule. Furthermore, when an antibody is further bound to the protein, competition between the labeling molecule and the antibody (e.g., competition due to multiple antibodies) can be reduced.

[0013] One aspect of the present disclosure is, for example, the following [4]: ​​[4] The evaluation method according to any one of [1] to [3], wherein the labeling step further comprises contacting the protein with an antibody or an antigen-binding fragment thereof that is capable of binding to the protein and that is modified with a third reporter enzyme, and the method comprises measuring an enzymatic reaction of the third reporter enzyme in a solution containing one molecule of the labeled protein.

[0014] According to the evaluation method described in [4], a protein in a solution can be detected using an enzymatic reaction of a third reporter enzyme, with a signal derived from a reaction product generated by the third reporter enzyme metabolizing a substrate as an indicator. This allows the enzymatic reaction of the third reporter enzyme to confirm that a single molecule of protein is present in the solution. For example, when proteins are arranged so that a single molecule of protein can be stochastically present in the solution, a solution containing a single molecule of protein can be selected from candidate solutions using the enzymatic reaction of the third reporter enzyme.

[0015] One aspect of the present disclosure is, for example, the following [5]: [5] The evaluation method according to any one of [1] to [4], wherein the labeling step further comprises contacting the complex of beads and an antibody or antigen-binding fragment thereof that binds to the protein with the protein so that one molecule of the protein binds to each complex, and the evaluation method further comprises, after the labeling step and before the detection step, placing the complex bound to the protein in microwells so that one bead in the complex is contained per well.

[0016] According to the evaluation method described in [5], due to the specificity of the antibody or antigen-binding fragment thereof that binds to the protein, evaluation can be performed under conditions where a solution containing a single molecule of the protein is a solution that selectively contains the protein to be evaluated (a solution containing only one molecule of the target protein as a biopolymer). Here, when a protein in a biological sample (such as a specimen) is the evaluation target, conventional evaluation methods using a single-molecule evaluation system prepared by stochastic diffusion of biopolymers have been unable to determine whether the protein in the single-molecule evaluation system is the evaluation target (e.g., whether it is the target isotype). In contrast, according to the evaluation method described in [5], the activity / reactivity of the active / reactive site can be evaluated under conditions that ensure that the protein in the single-molecule evaluation system is the evaluation target (target). Furthermore, according to the evaluation method described in [5], the protein bound to the antibody or antigen-binding fragment thereof is isolated from the reaction solution used in the labeling step by arranging beads, so the activity / reactivity of the active / reactive site can be evaluated under conditions where the protein is substantially concentrated. Therefore, when the target protein in a sample (such as a specimen) of biological origin is to be evaluated, the activity / reactivity of the active / reactive site can be evaluated even when the concentration of the target protein is several orders of magnitude lower than in conventional evaluation methods that use a single-molecule evaluation system prepared by the stochastic diffusion of biopolymers (e.g., the evaluation method described in Non-Patent Document 3) (i.e., the evaluation limit concentration of the target protein is several orders of magnitude lower).

[0017] One aspect of the present disclosure is, for example, the following [6]: [6] The evaluation method according to [5], wherein the beads are fluorescent.

[0018] According to the evaluation method described in [6], the presence or absence of beads can be evaluated based on fluorescence. As a result, for example, microwells in which fluorescence from beads is detected can be selected as wells to be evaluated. Furthermore, for example, according to this embodiment, microwells to be evaluated can be determined without using two or more antibodies that bind to a single enzyme, as in the digital ELISA method. This can reduce the risk of reduced evaluation accuracy due to, for example, antibody competition.

[0019] One aspect of the present disclosure is, for example, the following [7]. According to this aspect, an evaluation system containing one bead per well can be easily prepared. [7] The evaluation method according to [5] or [6], wherein the disposing step includes: suspending the complex bound to the protein into a plurality of storage compartments of a microchamber array having a hydrophobic surface; pouring an aqueous solution containing a substrate for the first reporter enzyme into the plurality of storage compartments through openings so that the number-average number of beads filled in each of the plurality of storage compartments is 0.0002 to 0.5; and blocking the openings with a hydrophobic solvent.

[0020] One aspect of the present disclosure is, for example, the following [8] or [9]. According to these aspects, a protein labeled with an activity-based labeling molecule can be simply modified with a first reporter enzyme. [8] The evaluation method according to any one of [1] to [7], wherein the activity-based labeling molecule includes a reactive site that reacts with the active / reactive site of the protein and a first labeling site, and the labeling step further includes contacting the activity-based labeling molecule with the protein and then directly or indirectly modifying the first labeling site derived from the activity-based labeling molecule with the first reporter enzyme. [9] The evaluation method according to any one of [1] to [8], wherein the activity-based labeled molecule comprises a reactive site that reacts with the active / reactive site of the protein and a first labeling site, the labeling step comprises contacting the protein with the activity-based labeled molecule and then further contacting the protein with a first reporter enzyme-labeled molecule, and the first reporter enzyme-labeled molecule is a molecule comprising a first binding site that specifically binds to the first labeling site of the activity-based labeled molecule and the first reporter enzyme modified thereto.

[0021] One aspect of the present disclosure is, for example, the following

[10] or

[11] . According to these aspects, a protein labeled with a post-translational modification labeling molecule can be simply modified with a second reporter enzyme.

[10] The evaluation method according to any one of [2] to [9], wherein the post-translational modification labeling molecule includes an interaction site that interacts with the post-translational modification site and a second labeling site, and the labeling step further includes contacting the protein with the post-translational modification labeling molecule, and then directly or indirectly modifying the second labeling site derived from the post-translational modification labeling molecule with the second reporter enzyme.

[11] The evaluation method according to any one of [2] to

[10] , wherein the post-translationally modified labeled molecule comprises an interaction site that interacts with the post-translation modification site and a second labeling site; the labeling step comprises contacting the protein with the post-translationally modified labeled molecule and then further contacting the protein with a second reporter enzyme-labeled molecule; and the second reporter enzyme-labeled molecule is a molecule comprising a second binding site that specifically binds to the second labeling site of the post-translationally modified labeled molecule and the second reporter enzyme modified thereto.

[0022] One aspect of the present disclosure is, for example, the following

[12] . According to this aspect, the activity / reactivity of the active center of an enzyme and / or the highly reactive residue of a protein can be evaluated.

[12] The evaluation method according to any one of [1] to

[11] , wherein the active / reactive site is the active center of an enzyme and / or the highly reactive residue of a protein.

[0023] The evaluation method according to the present disclosure enables highly sensitive evaluation of the activity / reactivity of an active / reactive site of a protein at the single-molecule level, and does not require the development of an activity detection probe that generates an increase in signal intensity due to the action of the protein for each protein to be detected.

[0024] 1 is a diagram showing an outline of an evaluation method according to a first embodiment; 2 is a diagram showing an outline of an evaluation method according to a second embodiment; 3 is a diagram showing the results of observing, in an oil-sealed chamber system, the fluorescence of a fluorescent dye produced by the metabolism of a reporter enzyme for beads capturing PSA whose reactive lysine residues have been modified with ALP; 4 is a diagram showing the results of evaluating, in an oil-sealed chamber system, the labeling of the active site of PSA with ABP, by SDS-PAGE and chemiluminescence; 5 is a diagram showing the results of observing, in an oil-sealed chamber system, the fluorescence of a fluorescent dye produced by the metabolism of a reporter enzyme for fluorescent beads capturing PSA whose active site serine residues have been modified with β-Gal; 6 is a diagram showing the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites was observed among wells in which green fluorescence derived from beads was observed in Example 2; 7 is a diagram showing the results of evaluating PSA activity based on the enzymatic activity of a reporter enzyme for Example 3; 8 is a diagram showing the results of evaluating PSA activity by detecting labeling with ABP using chemiluminescence for Example 3;

[0033] Figure 1 shows the results of measuring the amount of active PSA and the amount of total PSA in lysates of 22Rv1 cells and LNCap cells in Example 4.

[0034] Figure 1 shows the results of comparing the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites that modified active PSA was observed among wells in which green fluorescence derived from beads was observed, between different cell types and in the presence or absence of inhibitors in Example 4.

[0035] Figure 1 shows the value obtained by dividing the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites that modified active PSA was observed among wells in which green fluorescence derived from beads was observed by the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites that modified total PSA was observed among wells in which green fluorescence derived from beads was observed.

[0036] Figure 1 shows the increase in absorbance before and after incubation of lysates of 22Rv1 cells and LNCap cells in PSA activity measurement in a bulk system in Example 4.

[0037] Figure 1 shows the results of measuring the amount of active PSA and the amount of total PSA in plasma from healthy subjects in Example 5.1 shows the results of evaluating the activity of PSA when PSA was mixed with plasma derived from a healthy subject and then labeled with an activity-based labeling molecule (Pre-mix group) and when PSA was labeled with an activity-based labeling molecule and then mixed with plasma derived from a healthy subject (Post-mix group) in Example 6.

[0034] FIG. 1 shows the results of evaluating the activity of Granzyme B when a 0.1 μg / mL Granzyme B-azide solution was labeled with ABP in the same manner as in the preliminary study in Example 2, and then luminescence was detected by SDS-PAGE and chemiluminescence.

[0035] FIG. 1 shows the results of measuring the amount of active Granzyme B in a group to which a reagent required for the click reaction was added (Click+) and a group to which a reagent required for the click reaction was not added (Click-, control group) in Example 7. FIG. 10 is a diagram showing the percentage (%) of wells in which red fluorescence derived from an active metabolite of β-Gal modified with Granzyme B was observed among wells in which green fluorescence derived from beads was observed in the group (Click) to which a reagent necessary for the click reaction was added and the group (Control) to which a reagent not necessary for the click reaction was added in Example 7. FIG. 11 is a diagram showing the results of evaluating the activity of Granzyme B in liver injury model mice and healthy mice in Example 8. FIG. 11 is a diagram showing the percentage (%) of wells in which red fluorescence derived from an active metabolite of β-Gal modified with Granzyme B was observed among wells in which green fluorescence derived from beads was observed.

[0025] Modes for carrying out the present disclosure will be described below, but the present disclosure should not be construed as being limited to the following embodiments.

[0026] [Definitions and Terminology] In the present disclosure, "-" and "=" between two groups in a chemical formula indicate a single bond and a double bond, respectively.

[0027] The alkyl is a group obtained by removing one hydrogen atom from an alkane. The alkyl may be a group obtained by removing one hydrogen atom from a chain or cyclic alkane, and preferably a group obtained by removing one hydrogen atom from a chain alkane, and the chain alkane may be linear or branched. In one embodiment, the alkyl is １－５ It may be alkyl.１－５ Alkyl is an alkyl having from 1 to 5 carbon atoms. Alkyl may be, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, or cyclopentyl; １－５ It is alkyl.

[0028] Alkenyl is a group obtained by removing one hydrogen atom from an alkene. The alkenyl may be a group obtained by removing one hydrogen atom from a chain or cyclic alkene, and preferably a group obtained by removing one hydrogen atom from a chain alkene, and the chain alkene may be linear or branched. In one embodiment, the alkyl is C ２－４ It may be alkenyl. ２－４ Alkenyl is an alkenyl having from 2 to 4 carbon atoms. Alkenyl may be, for example, vinyl, allyl, 2-butenyl, or 3-butenyl, which are C ２－４ It is alkenyl.

[0029] An alkoxy is a monovalent group in which an alkyl is bonded to a root oxygen atom. In one embodiment, the alkoxy may be an alkoxy containing a linear alkyl. In one embodiment, the alkoxy may be C １－５ It may be alkoxy. １－５ Alkoxy is an alkoxy having from 1 to 5 carbon atoms. Alkoxy can be, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy or neopentoxy; １－５ In one aspect, the alkoxy may be methoxy, ethoxy, propoxy, butoxy, or pentoxy, which include linear alkyls. １－５ It is an alkoxy.

[0030] Alkoxymethyl is a group in which one hydrogen atom of methyl is substituted with alkoxy. In one embodiment, the alkoxymethyl may be a group in which one hydrogen atom of methyl is substituted with alkoxy containing a linear alkyl. In one embodiment, the alkoxymethyl is a group having C１－５ It may be alkoxymethyl. １－５ Alkoxymethyl is a group in which one hydrogen atom of methyl is substituted with alkoxy having 1 to 5 carbon atoms. Alkoxymethyl may be, for example, methoxymethyl, ethoxymethyl, propoxymethyl, butoxymethyl or pentoxymethyl, which are straight-chain C １－５ It is an alkoxymethyl containing alkyl.

[0031] Halogen is an element belonging to Group 17 of the periodic table, and in one embodiment may be fluorine, chlorine, bromine, or iodine. Halogeno is a monovalent group corresponding to halogen, and in one embodiment may be fluoro, chloro, bromo, or iodo.

[0032] A substituent is a monovalent group that replaces a hydrogen atom. In one embodiment, a substituent is a monovalent group that replaces a hydrogen atom on a carbon atom. An "optionally substituted" functional group may have no substituent, or may have one, two, three or more substituents, each of which is different. In one embodiment, an "optionally substituted" functional group may have no substituent or one substituent. In a preferred embodiment, an "optionally substituted" functional group may have no substituent. That is, in a preferred embodiment, an "optionally substituted" functional group may be the functional group itself.

[0033] In the present disclosure, the "substituent" may be, for example, but is not limited to, alkyl, alkenyl, alkoxy, alkoxymethyl, halogeno, amino group, phenyl, or carboxy, and these may further have a substituent.

[0034] In the present disclosure, a "monovalent organic group" may be, for example, a hydrogen atom, an alkyl, an alkenyl, an alkoxy, an alkoxymethyl, a halogeno, an amino group, a phenyl, or a carboxy.

[0035] <Activity-based protein profiling (ABPP)> The protein evaluation method according to the first embodiment of the present disclosure can be said to be an application of activity-based protein profiling (ABPP) to evaluating proteins at the single molecule level. In a more detailed aspect, the protein evaluation method according to the first embodiment of the present disclosure can also be an application of the ABPP method to a digital ELISA method. Therefore, ABPP and digital ELISA will first be described.

[0036] ABPP is a functional proteomics methodology. It uses activity-based probes (ABPs) that form covalent bonds depending on the specific function of a protein. When the activity-based probe is contacted with a protein, a covalent bond is formed between the functional protein and the activity-based probe, labeling the protein. The reporter moiety of the activity-based probe can then be used to perform proteome analysis. Activity-based probes contain at least a warhead moiety that forms a covalent bond with the protein and a reporter moiety. Examples of warhead moieties include suicide substrates that selectively bind to the active site of a specific enzyme, and electrophilic species that form covalent bonds with nucleophilic amino acid side chains (e.g., Lys, Cys). Examples of reporter moieties include fluorophores, which enable fluorescence detection; biotin, which enables avidin-based pull-down isolation or modification of other reporter molecules; and alkynes and azides, which enable click-reaction pull-down isolation or modification of other reporter molecules.

[0037] ABPPs and activity-based probes are described in more detail below. The concept of ABPPs has been known since the 1990s, but around 2000, it was reported that activity-based probes with fluorophosphonate warheads could broadly label the active sites of over 200 types of serine hydrolases (Non-Patent Document 4). Around the same time, it was also reported that activity-based probes with various electrophilic species warheads could label the active sites of cysteine ​​proteases (Non-Patent Document 5). For example, ABPPs that detect such active sites, when combined with mass spectrometry, are widely used in research to identify functional changes in proteins associated with diseases, etc., as a method that can simultaneously analyze the activities of multiple molecular species, including unknown proteins (Non-Patent Document 6). These early-developed ABPPs perform broad labeling using warheads that label the active sites of specific enzymes, enabling comprehensive activity profiling using platforms such as electrophoresis and mass spectrometry. To date, ABPPs using activity-based probes for a variety of enzyme groups, such as serine hydrolases, cysteine ​​proteases, metalloproteases, kinases, caspases, deubiquitinases, P450s, glycosidases, and phosphatases, have been reported (Non-Patent Document 7).

[0038] Meanwhile, as biological research into labeling and comprehensively analyzing proteins with specific functions, such as enzyme activity, has become more prevalent, the concept has been proposed that it is possible to read out changes in the reactivity of not only the active sites of enzymes, but also amino acids on the protein surface as functional information. The original research was initiated by a 2010 report that found that some cysteine ​​residues constituting proteins are highly reactive, and that these highly reactive cysteines are likely to play an important role in the function of proteins (the marked correlation between cysteine ​​hyper-reactivity and functionality) (Non-Patent Document 8). This report found a group of cysteines that exhibited particularly fast reaction rates when labeling protein cysteines with electrophilic species, and based on the following three facts (1) to (3), suggested that these cysteines may be a functionally important group. The mechanism of high reactivity is that the pK of the mercapto group of cysteine, which is normally around 8 to 9, is increased due to the influence of the side chains of the surrounding amino acids. ａ It is believed that this is due to the fact that the structure of cysteines has changed, resulting in high reactivity even under neutral conditions. (1) When compared with the UniProt database, it was found that many of the cysteines with literature reports on their functions contained functionally important sites such as enzyme activity and redox response. (2) The cysteines found to exhibit particularly fast reaction rates have a high proportion of those conserved throughout the evolution of mammals. (3) When cysteines with no reported functions were chemically modified or mutated in proteins, there were cases in which the protein lost its function.

[0039] Since the publication of Non-Patent Document 8, ABPP has further developed in terms of both its application and the range of reactive residues that can be labeled. For example, it has been developed as a method for identifying drug discovery targets. Specifically, the possibility of drug discovery targeting highly reactive cysteines has been proposed, and active efforts have been made to develop new drugs using methodologies that comprehensively evaluate the reactivity of cysteine ​​residues and their chemical modification by covalent inhibitors (Non-Patent Documents 9-11). In particular, in recent years, examples have been reported in which useful inhibitors can be developed for targets previously considered undruggable, such as K-Ras, by using covalent bonding with cysteine ​​as a foothold (Non-Patent Document 12). Thus, ABPP has developed into a methodology for broadly evaluating the potential for covalent bonding of active and reactive sites of proteins in the development of inhibitors via the formation of covalent bonds with target proteins. The range of reactive residues to be labeled has been expanded to include studies focusing on differences in the reactivity of lysine (Non-Patent Document 13) and differences in the environment of tyrosine (Non-Patent Document 14), based on the concept of broad profiling of the reactivity of amino acid side chains. Profiling studies using activity-based probes for these have also been conducted. As with the above-mentioned cysteine, some theoretical background has also been described for lysine (Non-Patent Document 13). Furthermore, the development of activity-based probes that can be used for other amino acid modifications is anticipated.

[0040] Based on the above, we will summarize ABPP. Early ABPP was ABPP in the narrow sense, which attempted to understand and evaluate enzyme activity as a function using activity-based probes that label the active site of an enzyme. On the other hand, recent ABPP is ABPP in the broad sense, which attempts to understand and evaluate changes in the overall function of a protein caused by differences in the reactivity of reactive amino acids using activity-based probes that label reactive amino acids such as cysteine, lysine, and tyrosine present on the protein surface. Both methods have in common the fact that the activity-based probe forms a covalent bond with the protein to be evaluated and that the labeling by the activity-based probe is detected to understand and evaluate the overall function of the protein.

[0041] <Digital ELISA> Digital ELISA is a method in which the reporter enzyme reaction in ELISA is carried out in a minute volume of solution that can contain a single molecule of protein. In conventional ELISA, reporter enzyme activity is detected in a solution in which a large number of target proteins may be present, and the protein is quantified using the amount of reaction product produced by the metabolic reaction of the reporter enzyme as an indicator. In contrast, in digital ELISA, each solution contains only one molecule of the target protein, and the reaction product produced by the metabolic reaction of the reporter enzyme can be sufficiently detected from the solution containing the target protein. Therefore, the measurement results for each solution are binarized as either "presence" or "absence" of the target protein. Therefore, in digital ELISA, an assay in a solution containing only one molecule of the target protein is performed on multiple solutions, and the protein is quantified using the proportion of solutions that are evaluated as "presence" of the target protein among the multiple solutions as an indicator. Digital ELISA requires the use of two antibodies that can simultaneously bind to one molecule of the protein to be evaluated, just like regular ELISA, but it can detect proteins with higher sensitivity than conventional ELISA.

[0042] <Method for Evaluating Activity / Reactivity of Active / Reactive Sites in Proteins> Based on the above, a method for evaluating the activity / reactivity of active / reactive sites in proteins according to a first embodiment of the present disclosure will be described. An outline of the evaluation method of this embodiment is shown in FIG. 1. In the evaluation method of this embodiment, a target protein is first labeled with an activity-based labeling molecule. Then, the activity-based labeling molecule, which has been further modified with a reporter enzyme, is detected at the single-molecule level based on the activity of the reporter enzyme.

[0043] The method for evaluating the activity / reactivity of an active / reactive site of a protein according to this embodiment will be described in more detail below. First, each element used in the evaluation method of this embodiment (target protein, activity-based labeled molecule, reporter enzyme, substrate, reporter enzyme-labeled molecule, etc.) will be described, followed by a description of the overall flow of the evaluation method and each step.

[0044] <Target Protein> The protein to be evaluated (target protein) in the evaluation method of this embodiment may be any protein that has or has the potential to have an active site and / or a reactive site. The target protein is not limited to proteins consisting only of amino acid residues. For example, the target protein may be a protein that has undergone post-translational modifications such as phosphorylation, glycosylation, or hypersulfur modification, or may be a protein that contains a metal ion as a cofactor (metalloprotein). The target protein may contain, for example, natural amino acids and unnatural amino acids as its amino acid residues, or may contain only natural amino acids. The target protein may contain, for example, L- and D-amino acids as its amino acid residues, or may contain only L-amino acids. The target protein may contain, for example, α-amino acids, β-amino acids, and γ-amino acids as its amino acid residues, or may contain α-amino acids and β-amino acids, or may contain only α-amino acids. In the present disclosure, proline is considered to be included in α-amino acids. The target protein may contain, for example, primary amino acids and secondary amino acids as its amino acid residues, or may contain primary amino acids and proline. The amino acids according to the present disclosure may be selected from the group consisting of, for example, alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). In the present disclosure, the amino acid sequences of peptides and proteins are written from the N-terminus to the C-terminus unless otherwise specified.

[0045] The protein of interest may have at least one active and / or reactive site, and may have two, three, four, five or more active and / or reactive sites, but in one aspect may have only one active and / or reactive site.

[0046] The active site of a protein of interest refers to a site that exhibits the function of changing the chemical structure of a substance under physiological conditions. In this case, the activity of the active site of a protein of interest refers to the function of the active site of the protein of interest changing the chemical structure of a substance. In other words, the active site of a protein of interest refers to a site that exhibits the function of metabolizing a substance under physiological conditions. In this case, the activity of the active site of a protein of interest refers to the function of the protein of interest metabolizing a substance. In one embodiment, the protein of interest may be an enzyme. In this case, the active site may be the active center of the enzyme, and the activity may be enzymatic activity. In addition, in this case, the substance whose chemical structure is changed by the active site of the protein of interest is a substrate of the enzyme. The target protein according to the evaluation method of this embodiment is not particularly limited, and may be, for example, an enzyme classified as EC3 (hydrolase), EC2 (transferase), or EC1 (oxidoreductase) in the EC7 EC classification established in 2019 by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), or may be an enzyme classified as EC3, or may be an enzyme classified as EC3.4 (peptidase), EC3.2 (glycosidase), or EC3.1 (esterase), or may be an enzyme classified as EC3.4, or may be an enzyme classified as EC3.4.21 (serine endopeptidase). The target protein in the evaluation method of this embodiment may be, for example, a serine hydrolase, a cysteine ​​protease, a metalloprotease, a kinase, a caspase, a deubiquitinase, a P450, a glycosidase, or a phosphatase; it may be a serine hydrolase, a cysteine ​​protease, a kinase, a phosphatase, or a glycosidase; it may be a peptidase, an exopeptidase, an endopeptidase, or a serine endopeptidase.Furthermore, the target protein according to the evaluation method of this embodiment may be, for example, an enzyme containing serine, cysteine, threonine, aspartic acid, or glutamic acid in its active site, or an enzyme containing serine in its active site. Examples of such enzymes include PSA (prostate specific antigen, also known as KLK3 or Kallikrein 3) and granzyme B.

[0047] A reactive site of a target protein refers to a site that reacts with a certain substance under physiological conditions, forming a covalent bond between the reactive site and that substance. In this case, the reactivity of a reactive site of a target protein refers to the function of the reactive site of the target protein forming a covalent bond with a certain substance. It is known that certain enzymes have amino acid residues in their active centers that are more active than normal amino acid residues, and that these activated residues first act on a substrate, causing a change in the chemical structure of the substrate. Therefore, a highly active amino acid residue present in the active center of such an enzyme can be considered a reactive site in addition to the active site described above, because it forms a covalent bond with the suicide substrate when contacted with the enzyme's suicide substrate. In one aspect, the reactive site of a target protein may be the active center of an enzyme having a highly active amino acid residue in its active center, or a highly reactive amino acid residue (highly reactive residue) on the surface of the protein. An amino acid residue on the surface of a protein refers to an amino acid residue exposed on the surface of the protein in a manner that allows it to be contacted by substances outside the protein. Whether a certain amino acid residue is exposed on the protein surface can be determined by referring to the three-dimensional structure determined by X-ray crystal structure analysis or cryo-electron microscopy, etc., contained in databases such as the RCSB Protein Data Bank (RCSB PDB). A highly reactive amino acid residue may be, for example, an amino acid residue having an amino, mercapto, hydroxy, or carboxy in the side chain. The reactivity of an amino acid side chain of a protein can change specifically depending on changes or modifications to the protein surface structure. A highly reactive amino acid residue is one that has a pK of the amino, mercapto, hydroxy, or carboxy in the side chain, compared to when it exists as a monoamino acid. ａThe highly reactive amino acid residue may be, but is not limited to, an amino acid residue whose β-labeling ratio varies by 0.3 or more, 0.5 or more, 1.0 or more, 1.5 or more, or 2.0 or more. Furthermore, the highly reactive amino acid residue may be, for example, lysine, cysteine, tyrosine, arginine, threonine, aspartic acid, or glutamic acid, or may be lysine or cysteine. In one aspect, the target protein in the evaluation method of this embodiment may be a protein having a highly reactive amino acid residue on its surface as described above. Such highly reactive amino acid residues are believed to be present on the surface of a wide variety of proteins. Examples of such target proteins include PSA, tau protein, CXCL11, PD-1, and PD-L1. Of the 227 residues in PSA, 8 are cysteine ​​residues. Furthermore, the present inventors demonstrated in the Examples herein that lysine residues on the surface of PSA can be labeled. Tau protein is the only amyloid protein containing cysteine. Furthermore, in relation to the labeling of post-translational modifications described below, it is known that the manner in which tau forms multimers differs depending on its phosphorylation state, and that these differences are important in neurodegenerative diseases. CXCL11 (C-X-C motif chemokine 11) has cysteine ​​residues on its surface. The four cysteine ​​residues present on the surface of CXCL11 form disulfide bonds, and these disulfide bonds are known to contribute to maintaining the protein structure. Furthermore, in relation to the labeling of post-translational modifications described below, it has been suggested that cysteines may be persulfidated by the action of extracellular glutathione persulfides. PD-1 (Programmed Cell Death 1) and PD-L1 (Programmed Death-Ligand 1) are therapeutic targets for anticancer drugs whose mechanism is to inhibit immune checkpoints. Therefore, if the target protein is PD-1 or PD-L1, detecting changes at the proteoform level may lead to the discovery of new therapeutic targets targeting PD-1 or PD-L1.

[0048] The target protein may be a protein contained in a sample or a purified protein, and in one embodiment, may be a protein contained in a sample. The sample may be derived from, for example, a human. The sample may be, for example, at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, tissue homogenates, cell homogenates, and extracts thereof, or at least one selected from the group consisting of plasma, serum, urine, saliva, tears, cerebrospinal fluid, and extracts thereof, or at least one selected from the group consisting of plasma, serum, tears, cerebrospinal fluid, and extracts thereof. When the target protein is a protein contained in a sample, the evaluation method according to the present disclosure can evaluate the sample itself, or can obtain information for diagnosing the animal (e.g., human) from which the sample was derived.

[0049] <Activity-based labeling molecule> An activity-based labeling molecule is a molecule that corresponds to the activity-based probe in ABPP. The activity-based labeling molecule has at least a reactive site. When the activity-based labeling molecule is contacted with a target protein, the reactive site forms a covalent bond with the activity and / or reactivity of the active site and / or reactive site of the target protein. In other words, the reactive site of the activity-based labeling molecule can be said to be a site that corresponds to the warhead site of the activity-based probe in ABPP.

[0050] The reactive site may be any site capable of forming a covalent bond with a target protein or its active site and / or reactive site. The reactive site may be, for example, a site that forms a direct covalent bond with a highly reactive amino acid residue present in the active center of an enzyme (i.e., a suicide substrate for the enzyme), an electrophilic species that forms a covalent bond with a nucleophilic group on an amino acid side chain, or a site that is metabolized by the enzyme and generates a highly reactive intermediate upon partial elimination, or may be an electrophilic species that forms a covalent bond with a suicide substrate for the enzyme or a nucleophilic group on an amino acid side chain. Such a reactive site may be, for example, a group that can be used as a warhead site for an activity-based probe in ABPP.

[0051] When the reactive site is an electrophilic species that forms a covalent bond with a suicide substrate of an enzyme or a nucleophilic group on an amino acid side chain, the reactive site may be, for example, a group described in Non-Patent Documents 4 to 14 or a group obtained by modifying such a group to the extent that the labeling ability is not impaired, or may be a group described in Non-Patent Documents 4 to 14. More specifically, for example, the reactive site may be a group selected from the group consisting of (A1) to (A9). (A1) A group represented by the following formula: [In the formula, R １ａ１ is a monovalent organic group, which in one embodiment may be alkoxy, for example, ethoxy.] (A2) A group represented by the following formula: [In the formula, R ２ａ１ is a monovalent organic group, which in one embodiment may be phenyl which may have a substituent, and in another embodiment is phenyl which is substituted with alkyl at the 2- and 6-positions and may further have substituents at the 3- to 5-positions, and may be, for example, 2,6-xylyl.] (A3) A group represented by the following formula: (A4) A group represented by the following formula: [In the formula, R ４ａ１ is a monovalent organic group, and in one embodiment is a group obtained by removing a hydrogen atom from the 5'-hydroxyl group of a nucleoside, and in another embodiment is a group obtained by removing a hydrogen atom from the 5'-hydroxyl group of a ribonucleoside, for example, a group obtained by removing a hydrogen atom from the 5'-hydroxyl group of adenosine.] (A5) A group obtained by replacing at least one secondary hydroxyl group of an aldose with fluoro and removing a hydrogen atom from a primary hydroxyl group. In one embodiment, this group may be a group obtained by replacing at least one secondary hydroxyl group of an aldohexose with fluoro and removing a hydrogen atom from a primary hydroxyl group. This group may be, for example, a group represented by the following formula: (A6) A group represented by the following formula: [In the formula, R ６ａ１ is a monovalent leaving group, which in one embodiment may be iodo, bromo, or chloro, for example, iodo.] (A7) A group represented by the following formula: (A8) A group represented by the following formula: [In the formula, R ８ａ１ is a monovalent organic group, which in one embodiment may be alkyl, for example, ethyl.８ａ２ , R ８ａ３ , R ８ａ４ and R ８ａ５ are each independently a monovalent organic group, and in one embodiment may each independently be a hydrogen atom, an alkyl, or an alkenyl, for example, a hydrogen atom.] (A9) A group represented by the following formula: [In the formula, R ９ａ１ is a monovalent organic group, and in one embodiment, it may be a phenyl group which may have a substituent, and in another embodiment, it is a phenyl group which is substituted with an alkoxy at the 4-position and may further have a substituent at the 2-, 3-, 5-, and 6-positions, and may be, for example, 4-methoxyphenyl. ９ａ２ , R ９ａ３ , R ９ａ４ and R ９ａ５ are each independently a monovalent organic group, and in one embodiment, may each independently be a hydrogen atom, an alkyl, or an alkenyl, for example, a hydrogen atom.

[0052] When the reactive site is a site that generates a highly reactive intermediate upon being metabolized by an enzyme and a portion thereof is eliminated, the highly reactive intermediate may be, for example, a quinone methide, a carbene, or a nitrene, and in one embodiment, may be a quinone methide. When the highly reactive intermediate is a quinone methide, the reactive site can be designed, for example, according to WO 2019 / 172210. Such a highly reactive intermediate can form a covalent bond with a target protein, for example, a residue contained in the active site or a residue present in the vicinity of the active site.

[0053] The activity-based labeling molecule may have a reporter enzyme or a labeling site in addition to the reactive site. These correspond to the reporter site in ABPP. From the viewpoint of convenience, the activity-based labeling molecule preferably has a labeling site in addition to the reactive site. Reporter enzymes will be described later.

[0054] The labeling site is a site that can be directly or indirectly modified with a reporter enzyme. The labeling site may be, for example, an affinity tag, an affinity tag protein, a click-reactive group, or an alkyl halide, and in one embodiment, may be an affinity tag or a click-reactive group. When the labeling site is an affinity tag or a click-reactive group, the binding between the affinity tag and the affinity tag protein and the click reaction tend to be highly orthogonal, making it easier to evaluate the activity / reactivity of the protein's active / reactive site.

[0055] In the present disclosure, an affinity tag and an affinity tag protein are a pair of a tag and a tag protein that form a highly specific bond. The bond formed may be a non-covalent bond or a covalent bond. The affinity tag and affinity tag protein according to the present disclosure may be any commonly used affinity tag and affinity tag protein known to those skilled in the art, and may be, for example, a biotin and a streptavidin, a Flag tag and a Flag tag protein, a Halo tag and a Halo tag protein, a SNAP tag and a SNAP tag protein, a Clip tag and a Clip tag protein, or a small molecule antigen and an antibody therefor.

[0056] In the present disclosure, a click-reactive group refers to a monovalent group that undergoes a click reaction, which is a chemical reaction applicable to click chemistry. In one aspect, the click-reactive group may be a monovalent group that undergoes a cycloaddition reaction applicable to click chemistry. In a preferred aspect, the click-reactive group may be a monovalent group that undergoes a metal ion-catalyzed or steric strain-promoted cycloaddition reaction. In a more preferred aspect, the click-reactive group may be a monovalent group that undergoes an azide-alkyne cycloaddition reaction or a strain-promoted inverse electron demand Diels-Alder reaction. In an even more preferred aspect, the click-reactive group may be a monovalent group that undergoes an azide-alkyne cycloaddition reaction from the viewpoint of the stability of the covalent bond that is formed.

[0057] (1) Azide-Alkyne Cycloaddition Reaction Azide and ethynylene undergo an azide-alkyne cycloaddition reaction, which is a 1,3-dipolar cycloaddition reaction, to form a 1,2,3-triazole ring. In one embodiment, the click-reactive group may be a group containing azide or ethynylene. The azide-alkyne cycloaddition reaction is known as a representative reaction in click chemistry, and the structure of the click-reactive group and the reaction conditions can be appropriately determined by those skilled in the art. It is known that the reaction conditions for the azide-alkyne cycloaddition reaction vary depending on the chemical structure of the ethynylene. The azide-alkyne cycloaddition reaction may be, for example, a Huisgen cyclization reaction or a strain-promoted alkyne-azide cycloaddition reaction (SPAAC).

[0058] (1-1) Huisgen Cyclization Reaction (CuAAC, Copper-Catalyzed Azide-Alkyne Cycloaddition) The Huisgen cyclization reaction is a cycloaddition reaction catalyzed by copper(I) ions, and is known to have a high tolerance for ethynylene structures. That is, in one embodiment, the click-reactive group may be a group containing azide or ethynylene, and in this case, the modification step may form a covalent bond by a 1,3-dipolar cycloaddition reaction catalyzed by copper(I) ions.

[0059] (1-2) Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) SPAAC is an azide-alkyne cycloaddition reaction that occurs between an azide and a strained ethynylene. In SPAAC, the ethynylene is sterically strained and in a high-energy state, allowing the azide-alkyne cycloaddition reaction to occur in the absence of a metal catalyst. SPAAC is known as a type of so-called copper-free click reaction. That is, in one embodiment, the click-reactive group may be a group containing an azide or a strained (highly strained) ethynylene. In this case, a covalent bond may be formed in the modification step in the absence of a metal catalyst, and the covalent bond is formed by a 1,3-dipolar cycloaddition reaction.

[0060] The group containing strained ethynylene is not particularly limited as long as it is known as a group applicable to SPAAC, and may be, for example, a group in which one hydrogen atom has been removed from cyclooctyne, dibenzocyclooctyne (DBCO), or bicyclo[6.1.0]nonyne (BCN), which may have a substituent, and one or two carbon atoms forming the ring may be substituted with a nitrogen atom.

[0061] (2) Strain-Promoted Inverse Electron-Demand Diels-Alder Cycloaddition (SPIEDAC) SPIEDAC is an inverse electron-demand Diels-Alder reaction that occurs between an electron-deficient heterocycle and a sterically strained dienophile. SPIEDAC is known as a type of copper-free click reaction because it occurs without a metal ion catalyst such as copper ion and also occurs under physiological conditions such as in water and at room temperature. That is, in one embodiment, the click-reactive group may be a group containing an electron-deficient heterocycle or a group containing a sterically strained (highly strained) dienophile. In this case, a covalent bond may be formed in the modification step in the absence of a metal catalyst, and the covalent bond is formed by an inverse electron-demand Diels-Alder reaction.

[0062] The group containing an electron-deficient heterocycle is not particularly limited as long as it is known as a group applicable to SPIEDAC, and may be, for example, a group described in the literature (BL Oliveira et al., "Inverse electron demand Diels-Alder reactions in chemical biology", Chem. Soc. Rev., 2017, 46, 4895-4950, Aysun Degirmenci et al., "Metal-Free Click-Chemistry: A Powerful Tool for Fabricating Hydrogels for Biomedical Applications", Bioconjugate Chemistry 2024, 35, 4, 433-452). The group containing an electron-deficient heterocycle may be, for example, a monovalent group containing a tetrazine ring or a triazine ring, and in one embodiment, it may be a monovalent group containing a 1,2,4,5-tetrazine ring. In a preferred embodiment, it may be a monovalent group obtained by removing a hydrogen atom from a compound in which at least one hydrogen atom of 1,2,4,5-tetrazine is substituted with a substituent. In a more preferred embodiment, it may be a group obtained by removing one hydrogen atom from a compound in which one hydrogen atom of 1,2,4,5-tetrazine is substituted with an aromatic ring compound (e.g., benzene, pyridine, or pyrimidine) which may have a substituent or an electron-withdrawing group (e.g., trifluoromethyl or methoxycarbonyl) and the other hydrogen atom is substituted with a substituent, or a group obtained by removing one hydrogen atom from an aromatic ring compound in which both hydrogen atoms of 1,2,4,5-tetrazine are each independently substituted with a substituent or an electron-withdrawing group.

[0063] The group containing a sterically strained dienophile is not particularly limited as long as it is known as a group applicable to SPIEDAC, and may be, for example, a group in which one hydrogen atom has been removed from trans-cyclooctene (TCO), bicyclo[6.1.0]nonyne (BCN), cyclooctyne, dibenzocyclooctyne (DBCO), or norbornene, or in one embodiment, a group in which one hydrogen atom has been removed from TCO or BCN, which may have a substituent, and one or two carbon atoms forming the ring may be substituted with nitrogen atoms.

[0064] The activity-based labeling molecule may further have a linker site. In one embodiment, the activity-based labeling molecule may include a reactive site and a reporter enzyme or labeling site, or may include a reactive site, a linker site, and a reporter enzyme or labeling site. However, from the viewpoint of achieving both labeling and modification, it is preferable that the activity-based labeling molecule include a reactive site, a linker site, and a labeling site. In this case, when the activity-based labeling molecule has a linker site, the reactive site and the labeling site form a conjugate molecule via the linker site.

[0065] When the activity-based labeling molecule includes a linker moiety, the chemical structure of the linker is not particularly limited as long as it can be used to form a conjugate molecule. The linker moiety may, for example, have one or more, two or more, three or more, four or more, five or more, seven or more, or ten or more atoms, or may have 1,000 or fewer, 500 or fewer, 200 or fewer, 50 or fewer, or 30 or fewer atoms, and these upper and lower limits may be freely combined. The linker moiety may, for example, have one or more, two or more, three or more, four or more, five or more, seven or more, or ten or more atoms, or may have 200 or fewer, 50 or fewer, 30 or fewer, 12 or fewer, 8 or fewer, 6 or fewer, or 5 or fewer atoms, and these upper and lower limits may be freely combined. The linker may be, for example, a linker consisting of at least one atom selected from the group consisting of a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, and a sulfur atom. The linker may also be, for example, a linker consisting of an aliphatic hydrocarbon, a peptide linker, a polyethylene glycol (PEG) linker, or a linker in which multiple linkers selected from the group consisting of these are linked via an amide bond, an ether bond, an ester bond, or a triazole bond.

[0066] <Reporter Enzyme / Substrate> The reporter enzyme according to the present disclosure is an enzyme that can evaluate the presence or absence of modification by the reporter enzyme by contacting it with a substrate and detecting the reaction product. The reporter enzyme may be a reporter enzyme that can detect the enzymatic reaction product, for example, by fluorescence measurement, Raman spectroscopy, or luminescence measurement. In one embodiment, it may be a reporter enzyme that can be detected by fluorescence measurement. Furthermore, when a single target protein molecule is modified with multiple types of reporter enzymes using the evaluation method according to the present disclosure, the substrate and reporter enzyme are preferably selected so that the signals from the reaction products generated by each type of reporter enzyme can be distinguished from each other. In detection by fluorescence measurement or luminescence measurement, the substrate and reporter enzyme may be selected, for example, so that the wavelength and / or signal lifetime are distinguishable from each other. In detection by Raman spectroscopy, the substrate and reporter enzyme may be selected, for example, so that the Raman shifts are distinguishable from each other.

[0067] The reporter enzyme and its substrate can be a known pair that has high specificity and produces a reaction product with a greater signal intensity than the substrate.

[0068] The reporter enzyme may be, for example, a peptidase, phosphatase, glycosidase, or aminoacyltransferase. The peptidase may be, for example, an exopeptidase or an endopeptidase. When the enzyme is a peptidase or aminoacyltransferase, the substrate may be a substance that increases signal intensity by hydrolysis, which removes an amino acid or peptide. When the enzyme is a phosphatase, the substrate may be a substance that increases signal intensity by hydrolysis, which cleaves a phosphate ester. When the enzyme is a glycosidase, the substrate may be a substance that increases signal intensity by hydrolysis, which cleaves a glycosidic bond. Suitable reporter enzymes include alkaline phosphatase (ALP), β-galactosidase (β-Gal), acylpeptide enzyme hydrolase (APEH), β-glucosidase Td2F2 (Td2F2), aminopeptidase N (CD13), Tobacco Etch Virus Protease (TEV protease), Ectonucleotide Pyrophosphatase / Phosphodiesterase 3 (ENPP3), γ-glutamyltransferase 1 (GGT1), pyroglutamate aminopeptidase from Pyrococcus furiosus, and aminopeptidase I from Pyrococcus furiosus (e.g., Takara Bio, Code No. 7336), pyroglutamyl aminopeptidase (pGluAP), acylamino-acid-releasing enzyme (AARE), and ectonucleotide pyrophosphatase / phosphodiesterase 6 (ENPP6).

[0069] The substrate of the reporter enzyme is not particularly limited, but may be composed of, for example, a monovalent cleavage site that is removed by cleavage by the reporter enzyme, and a fluorescence enhancement site (FIM) that generates or releases a fluorescent dye whose fluorescence intensity increases after removal of the cleavage site compared to before removal. The monovalent cleavage site and FIM are not particularly limited as long as they are used in fluorescent probes for detecting enzyme activity, but a non-restrictive example will be described below.

[0070] The monovalent cleavage site may be one that is removed by cleavage by the reporter enzyme, and can be appropriately selected by those skilled in the art depending on the reporter enzyme. For example, when the reporter enzyme is a peptidase, the monovalent cleavage site may be an amino acid or peptide that is recognized by the peptidase and cleaved at the N-terminus or C-terminus. In particular, when the reporter enzyme is APEH or AARE, the monovalent cleavage site may be an α-amino acid (e.g., methionine) in which the amino (α-amino group) bound to the α-carbon is acylated (e.g., acetylated), and the carboxy (α-carboxy group) bound to the α-carbon may form an amide bond with the amino of FIM. For example, when the reporter enzyme is CD13, the monovalent cleavage site may be an amino acid such as Ala, Arg, Met, or Tyr, and the α-carboxy group of these amino acids may form an amide bond with the amino acid of FIM (Anna Byzia et al., "Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library", Biochimie. 2016; 122:31-37). For example, when the reporter enzyme is TEV protease, the monovalent cleavage site may be ENLYFQG or ENLYFQS, and the α-carboxy group of the C-terminal amino acid may form an amide bond with the amino acid of FIM. For example, when the reporter enzyme is pyroglutamate aminopeptidase derived from Pyrococcus furiosus or pGluAP, the monovalent cleavage site may be pyroglutamic acid, and the α-carboxy group of the C-terminal amino acid may form an amide bond with the amino acid of FIM. For example, when the reporter enzyme is aminopeptidase I from Pyrococcus furiosus, the monovalent cleavage site may be an amino acid such as Leu, the α-carboxy group of which may form an amide bond with the amino acid of FIM. For example, when the reporter enzyme is a phosphatase, the monovalent cleavage site may be a methylene modified with a phospho or nucleotide together with an oxygen atom of FIM.In particular, for example, when the reporter enzyme is ALP, the monovalent cleavage site may combine with an oxygen atom of FIM to form a phospho or may be a methylene modified at a phospho. For example, when the reporter enzyme is ENPP3, the monovalent cleavage site may combine with an oxygen atom of FIM to form thymidine monophosphate (dTMP) or deoxyuridine monophosphate (dUMP), or may be a methylene modified at a phospho with dTMP or dUMP. For example, when the reporter enzyme is ENPP6, the monovalent cleavage site may combine with an oxygen atom of FIM to form a phosphocholine or may be a methylene modified at a phospho with phosphocholine. For example, when the reporter enzyme is a glycosidase, the monovalent cleavage site may be a sugar or a sugar chain. In detail, for example, when the reporter enzyme is β-Gal, the monovalent cleavage site may be D-galactose, and the hydroxyl on the carbon atom at position 1 may form an ether bond with FIM so that it is in the same spatial position as that of β-galactoside. For example, when the reporter enzyme is Td2F2, the monovalent cleavage site may be D-fucose, and the hydroxyl on the carbon atom at position 1 may form an ether bond with FIM so that it is in the same spatial position as that of α-fucoside. For example, when the reporter enzyme is aminoacyltransferase, the monovalent cleavage site may be an amino acid that is removed from the substrate of the reporter enzyme by rearrangement by the aminoacyltransferase. For example, when the reporter enzyme is GGT1, the monovalent cleavage site may be glutamic acid, and the carboxyl bonded to the γ-carbon may form an amide bond with the amino acid of FIM.

[0071] In one embodiment, the FIM is represented by FG-Y, where FG is a monovalent fluorescent group. １ -* or FG-Y ２ -BN-Y １ -*, where * represents a bond to the monovalent cleavage site. １ and Y ２ are each independently an oxygen atom or N(-R ２ ), where R ２ is a hydrogen atom, C １－６Alkyl or C ２－６ represents alkenyl, preferably a hydrogen atom or C １－６ It may represent alkyl, more preferably a hydrogen atom, methyl or ethyl, and even more preferably a hydrogen atom. In these cases, when the monovalent cleavage site is cleaved by the reporter enzyme, the reaction product is FG-Y ２ A fluorescent dye represented by -H (H is a hydrogen atom) is released.

[0072] Y ２ is an oxygen atom, BN may have a substituent on the benzene ring, and is represented by the following formula: [wherein * represents Y ２ The wavy line indicates the bond with Y １ In one embodiment, BN may represent a group represented by the same formula (i.e., a group having no substituent on the benzene ring in the above formula).

[0073] Y ２ is N(-R ２ ), BN may have a substituent on the benzene ring, and is represented by the following formula: [wherein * represents Y ２ The wavy line indicates the bond with Y １ In one embodiment, BN may represent a group represented by the same formula (i.e., a group having no substituent on the benzene ring in the above formula).

[0074] In one embodiment, FG may be a group represented by formula (III), formula (IV) or formula (V), may be a group represented by formula (III) or formula (IV), or may be a group represented by formula (IV). Hereinafter, "water-soluble substituent" refers to -CO ２ H, -PO ３ H ２ and -SO ３ H.

[0075] Formula (III) is as follows: The wavy line represents Y １ or Y ２In this case, the compound represented by FG-OH is a derivative of umbelliferone, a fluorescent dye. ２ The compound represented by formula (III)-H is a derivative of 7-aminocoumarin, a fluorescent dye. When FG is a group represented by formula (III), the molecule represented by formula (III)-FG-OH or FG-YH is a derivative of Y １ or Y ２ When the oxygen atom is not substituted, the absorption wavelength and fluorescence wavelength tend to be longer and the fluorescence quantum yield also tends to be higher, compared to when the hydrogen atom is substituted. Detailed design and organic synthesis of such FGs can be performed with reference to knowledge disclosed in publicly known literature.

[0076] R ａ１ ~R ａ５ are each independently a hydrogen atom; １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atom; nitro; hydroxy; hydroxy C which may have a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ alkoxy, where R ａ１ ~R ａ５ At least one of the C groups is a water-soluble substituent or a C group substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl or C １－６ It is an alkoxy.

[0077] In one embodiment, the group of formula (III) is R ａ４ is carboxy or a protected form thereof, and R ａ１ ~R ａ５３ and R ａ５ each independently represents a hydrogen atom; １－６ Alkyl, C ２－６ Alkenyl and C １－６Alkoxy; halogen atom; nitro; hydroxy; hydroxy C which may have a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ In another embodiment, the group represented by formula (III) may be a group selected from the group consisting of R ａ４ is carboxy or a protected form thereof, and R ａ１ ~R ａ５３ and R ａ５ each independently represents a hydrogen atom; １－６ Alkyl, C ２－６ Alkenyl and C １－６ Alkoxy; halogen atom; nitro; hydroxy and hydroxy C which may have a substituent on the carbon atom １－３ In a preferred embodiment, the group represented by formula (III) is R ａ４ is carboxy or a protected form thereof, and R ａ１ ~R ａ５３ and R ａ５ are each independently a hydrogen atom; C １－６ Alkyl; C ２－６ Alkenyl; C １－６ Alkoxy; halogen atom; C １－６ Haloalkyl; Nitro; Hydroxy; and HydroxyC １－３ In one particular embodiment, the group represented by formula (III) may be a group selected from the group consisting of R ａ４ is carboxy or a protected form thereof, and R ａ１ ~R ａ５３ and R ａ５or a group in which the phenolic hydroxyl group is removed from HCCA (Toru Komatsu et al., "Design and Synthesis of an Enzyme Activity-Based Labeling Molecule with Fluorescence Spectral Change", J. Am. Chem. Soc. 2006, 128, 50, 15946-15947).

[0078] Formula (IV) is as follows: The wavy line represents Y １ or Y ２ When FG is a group represented by formula (IV), a molecule represented by FG-OH or FG-YH can be bonded to Y by, for example, the following two principles: １ or Y ２ When the oxygen atom is not substituted, the fluorescence intensity is greater than when the hydrogen atom is substituted. (Principle A) Y １ or Y ２ When the hydrogen atom above is substituted, photoinduced electron transfer (PeT) occurs between SRG and the tricyclic moiety, resulting in a low fluorescence quantum yield. On the other hand, when the oxygen atom is not substituted, photoinduced electron transfer (PeT) does not occur, resulting in a high fluorescence quantum yield. (Principle B) Y １ or Y ２ When the above hydrogen atom is substituted, the intramolecular nucleophilic species of SRG is relatively favorably cyclized with the carbon atom to which SRG is bonded in formula (IV), which tends to disrupt a portion of the conjugated system of the tricyclic moiety, resulting in short absorption and fluorescence wavelengths and a low fluorescence quantum yield, whereas when the oxygen atom is not substituted, the intramolecular cyclization is relatively unfavorable, resulting in long absorption and fluorescence wavelengths and a high fluorescence quantum yield. The detailed design and organic synthesis of such FGs can be carried out with reference to knowledge disclosed in publicly known literature.

[0079] In formula (IV), SRG is a group represented by any of the following structural formulas:

[0080] Rｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ are each independently a hydrogen atom; １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C optionally having a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ alkoxy, with the proviso that R ｂ１ ~R ｂ５ and R ｃ１ ~R ｃ６ at least one of which is a water-soluble substituent; or C which is substituted with a water-soluble substituent and may further have a substituent １－６ Alkyl, C ２－６ Alkenyl or C １－６ It is an alkoxy.

[0081] In one embodiment, R ｂ１ ~R ｂ５ are each independently a hydrogen atom; １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C optionally having a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ alkoxy, with the proviso that R ｂ１ ~R ｂ５ at least one of which is a water-soluble substituent; or C which is substituted with a water-soluble substituent and may further have a substituent １－６ Alkyl, C ２－６ Alkenyl or C １－６ is alkoxy, and R ｃ１ ~R ｃ６ are each independently a hydrogen atom; １－６Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C optionally having a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ In one preferred embodiment, R ｂ１ ~R ｂ５ are each independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C １－３ is selected from the group consisting of alkyl and water-soluble substituents, with the proviso that R ｂ１ ~R ｂ５ at least one of which is a water-soluble substituent; or C substituted with a water-soluble substituent １－６ Alkyl, C ２－６ Alkenyl or C １－６ is alkoxy, and R ｃ１ ~R ｃ６ are each independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C １－３ In a more preferred embodiment, R ｂ１ ~R ｂ５ At least one is -SO ３ H, and the remainders are each independently a hydrogen atom, C １－６ Alkyl, C １－６ Alkoxy, hydroxy C １－３ is selected from the group consisting of alkyl and water-soluble substituents; R ｃ１ ~R ｃ６ are each independently a hydrogen atom, C １－６ Alkyl, C １－６ It is selected from the group consisting of alkoxy and halogen atoms.

[0082] In one embodiment, Rｄ１ and R ｄ２ each independently represents a hydrogen atom, a C １－６ Alkyl or C ２－６ C substituted with an alkenyl or water-soluble substituent and optionally further having a substituent １－６ Alkyl or C ２－６ alkenyl, where R ｄ１ and R ｄ２ are united in R ｄ１ and R ｄ２ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R is bonded, which may have a substituent, ｄ１ and / or R ｄ２ are respectively R ｃ５ or R ｃ６ Together with R ｄ１ or R ｄ２ may form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R is attached, which may have a substituent. ｄ１ and R ｄ２ are each independently a hydrogen atom, C １－６ Alkyl, C ２－６ C substituted with alkenyl or water-soluble substituents １－６ Alkyl or C ２－６ alkenyl, where R ｄ１ and R ｄ２ are united in R ｄ１ and R ｄ２ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R is bonded, ｄ１ and / or R ｄ２ are respectively R ｃ５ or R ｃ６ Together with R ｄ１ or R ｄ２ may form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which it is attached.

[0083] In one embodiment, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-Rｆ１ ) (-R ｆ２ ), where R ｆ１ and R ｆ２ each independently may have a substituent, C １－６ Alkyl, C ２－６ Alkenyl, C １－６ It may represent alkoxy or aryl. In one preferred embodiment, R ｅ is an oxygen atom, Si(-R ｆ１ ) (-R ｆ２ ) or C(-R ｆ１ ) (-R ｆ２ ), where R ｆ１ and R ｆ２ are each independently C １－６ Alkyl, C ２－６ Alkenyl, C １－６ It may represent alkoxy or aryl.

[0084] In a further preferred embodiment, the group represented by formula (IV) may be any of the groups represented by the following structural formulas:

[0085] In a specific embodiment, the group represented by formula (IV) may be any of the groups represented by the following structural formulas: These groups correspond to the groups obtained by removing a hydrogen atom from the hydroxy of sTM and sTG described in Non-Patent Document 3.

[0086] Formula (V) is as follows: The wavy line represents Y １ or Y ２ In this case, the compound represented by FG-OH is a derivative of resorufin, a fluorescent dye. When FG is a group represented by formula (III), the molecule represented by FG-OH or FG-YH is a bond to Y. １ or Y ２ When the oxygen atom is not substituted, the absorption wavelength and fluorescence wavelength tend to be longer and the fluorescence quantum yield also tends to be higher, compared to when the hydrogen atom is substituted. Detailed design and organic synthesis of such FGs can be performed with reference to knowledge disclosed in publicly known literature.

[0087] In one embodiment, Rｇ１ ~R ｇ６ are each independently a hydrogen atom; １－６ Alkyl, C １－６ Alkoxy and C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C optionally having a substituent on the carbon atom １－３ alkyl; a water-soluble substituent; and a C substituted with a water-soluble substituent and optionally further having a substituent. １－６ Alkyl, C ２－６ Alkenyl and C １－６ In one preferred embodiment, R ｇ１ ~R ｇ６ are each independently a hydrogen atom; C １－６ Alkyl; C １－６ Alkoxy; C ２－６ Alkenyl; halogen atom; nitro; hydroxy; hydroxy C １－３ In one particular embodiment, R ｇ１ ~R ｇ６ may represent a hydrogen atom. In this case, the group represented by formula (V) is a group obtained by removing hydroxy from resorufin. In one embodiment, FG may be a group represented by formula (III), formula (IV) or formula (V), may be a group represented by formula (III) or formula (IV), or may be a group represented by formula (IV).

[0088] <Reporter enzyme-labeled molecule> A reporter enzyme-labeled molecule is a molecule in which a target protein is labeled with an activity-based labeling molecule, and then the labeling site derived from the activity-based labeling molecule is directly or indirectly modified with a reporter enzyme. The reporter enzyme can be any of those described above as a <reporter enzyme>. A reporter enzyme-labeled molecule has a reporter enzyme and a binding site. Furthermore, a reporter enzyme-labeled molecule may have a linker site between the reporter enzyme and the binding site, and the linker site can be any of those described above for the linker site of the activity-based labeling molecule.

[0089] In one embodiment, the binding site may be a site that specifically binds to the labeling site of the activity-based labeling molecule. In this case, the binding site may have a structure that specifically binds to the labeling site of the activity-based labeling molecule. In this embodiment, the labeling site and the reporter enzyme-labeled molecule are directly bound to each other, thereby directly modifying the reporter enzyme at the labeling site.

[0090] In another embodiment, the binding site may be one of a pair that is different from the labeling site and the site that specifically binds thereto of the activity-based labeling molecule and that binds orthogonally to these bonds. In this case, the pair may be a specifically binding combination as described for the labeling site of the activity-based labeling molecule. In this case, modification of the target protein with the reporter enzyme is carried out in the following two steps. In the first step, the labeling site derived from the activity-based labeling molecule binds to the labeling site binding site of a conjugate molecule that has a site that specifically binds to the labeling site (labeling site binding site) and a site that specifically binds to the binding site (binding site binding site). In the second step, the binding site binding site derived from the conjugate molecule binds to the binding site of the reporter enzyme-labeled molecule. This results in one reporter enzyme being indirectly modified to the labeling site via the conjugate molecule. In this embodiment, the reporter enzyme is indirectly modified to the labeling site via the conjugate molecule. Note that the number of conjugate molecules in this embodiment is not limited to one; the reporter enzyme may be modified to the labeling site via two, three, four, five, or more conjugate molecules. Furthermore, the conjugated molecule of this embodiment may further include a linker site between the labeling site binding site and the binding site binding site, and the linker site may be one described for the linker site of the activity-based labeling molecule.

[0091] The reporter enzyme-labeled molecule can be prepared according to a method used to prepare a conjugate of an enzyme and a molecular compound, for example, by attaching (modifying) a binding site or a linker site to an enzyme. Specific methods for attaching a binding site or a linker site to an enzyme include, for example, (1) a method in which a carboxyl group in the binding site or linker site is converted into an active ester, and then an amide bond is formed between the active ester and an amino group in the enzyme, (2) a method in which a formyl group in the binding site or linker site is reductively bonded to an amino group in the enzyme, and (3) a method in which a maleimide group in the binding site or linker site is formed and a mercapto group in the enzyme, and a thioether bond is formed.

[0092] <Evaluation Example: Evaluation of Activity / Reactivity of Active / Reactive Site of Protein> Based on the above, the overall flow and each step of the method for evaluating the activity / reactivity of an active / reactive site of a protein according to the first embodiment will be described using a specific example. The following evaluation example includes, in this order, a step of labeling a target protein with an activity-based labeling molecule (labeling step), a step of modifying the target protein labeled with the activity-based labeling molecule with a reporter enzyme (modification step), a step of preparing a solution containing one molecule of the target protein modified with the reporter enzyme and a substrate for the reporter enzyme (preparation step), and a step of detecting the protein in the solution obtained in the preparation step using an enzymatic reaction of the reporter enzyme (detection step).

[0093] The evaluation system in the evaluation method according to the present disclosure is not particularly limited as long as it can form a closed reaction system consisting of one molecule of a target protein modified with a reporter enzyme and an aqueous solution containing a substrate for the reporter enzyme, and can measure a signal derived from the reaction product generated within the closed reaction system. The closed reaction system may be spatially enclosed, for example, by a resin or oil, and the volume of the closed reaction system may be, for example, 5 fL to 100 fL. Alternatively, multiple closed reaction systems may exist, and the detection step may be carried out within each of them. A more specific evaluation system may be, for example, an evaluation system using a microdevice with wells each having a volume of approximately 5 fL to 100 fL. In this case, the surface of the aqueous solution (closed reaction system) in the detection step that is not covered by the wells (openings) may be covered by an organic solvent or a gas (e.g., air). When the surface of the aqueous solution in the detection step that is not covered by the wells is covered by gas, the aqueous solution may remain in the wells due to, for example, gravity and / or surface tension. Another specific evaluation system may be, for example, an evaluation system using droplets (e.g., liposomes) present in an aqueous solution, each with a volume of approximately 5 fL to 100 fL. In this case, each droplet contains one molecule of a target protein modified with a reporter enzyme and an aqueous solution containing a substrate for the reporter enzyme, thereby enabling the enzymatic activity of each peptidase to be evaluated.

[0094] In a preferred embodiment, the evaluation system used in the evaluation method according to the present disclosure is an evaluation system using a microdevice equipped with wells, each with a volume of approximately 5 fL to 100 fL. In the detection step, the surface of the aqueous solution (closed reaction system) not covered by the wells may be covered with an organic solvent. Hereinafter, this evaluation system will be referred to as an "oil-sealed chamber system." Such evaluation systems are described, for example, in Patent Document 1 and Non-Patent Document 15. In an oil-sealed chamber system, a closed reaction system can be realized by utilizing the immiscibility of the hydrophobic solvent and the aqueous solution. The hydrophobic solvent is not particularly limited as long as it is immiscible with the aqueous solution. Suitable examples include at least one solvent selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, and hydrophobic ionic liquids, or a mixture containing such a solvent. As described above, a solution containing one molecule of a protein according to the evaluation method of the present disclosure can be realized, for example, by using a microdevice. As a specific example, the solution can be realized by arranging each well of a microchamber device, each droplet on a substrate, each liposome in an aqueous solution, or the like so that one molecule of the protein can be contained therein.

[0095] <Labeling Step> In the labeling step, the target protein is labeled with an activity-based labeling molecule. The labeling method is not particularly limited, and may be performed, for example, by contacting the target protein with the activity-based labeling molecule in a solvent. The solvent in this case may be any solvent that allows the target protein to be labeled with the activity-based labeling molecule, and may be, for example, an aqueous solvent or a buffer solution of pH 6 to 9.

[0096] The concentrations of the target protein and the activity-based labeling molecule and their relationship in the labeling step are not particularly limited, but it is preferable that the activity-based labeling molecule be in excess of the active / reactive site to be labeled in the target protein. In this case, the active / reactive site that should theoretically be labeled is sufficiently labeled, so that the activity / reactivity of the active / reactive site can be evaluated while suppressing the influence of labeling efficiency. In this respect, the activity-based labeling molecule may be added in an amount, for example, greater than 1 molar equivalent, 3 molar equivalents or more, 10 molar equivalents or more, 25 molar equivalents or more, or 50 molar equivalents or more of the target protein, or in an amount less than 1000 molar equivalents or less than 100 molar equivalents, and these upper and lower limits may be freely combined.

[0097] The contact conditions between the target protein and the activity-based labeling molecule in the labeling step are not particularly limited, as long as labeling occurs without denaturing the target protein. The contact temperature may be, for example, 4°C or higher and 50°C or lower, and examples thereof include 25°C, 37°C, and room temperature. The contact time may be, for example, 1 minute to 168 hours or 10 minutes to 24 hours, and examples thereof include 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, and 12 hours. The contact may be performed, for example, in an open, dark place. The contact may be performed, for example, while shaking or stirring.

[0098] The labeling step may include contacting the target protein with the activity-based labeling molecule, followed by ultrafiltration to collect the protein components. In this case, the target protein labeled with the activity-based labeling molecule can be collected while removing the activity-based labeling molecule that did not label the target protein as a filtrate. Filters that are sometimes used for ultrafiltration can be used, and a specific example is the Amicon (registered trademark) series.

[0099] <Modification step> In the modification step, a target protein labeled with an activity-based labeling molecule is modified with a reporter enzyme. When no conjugation molecule is involved, the modification step involves first labeling the target protein with an activity-based labeling molecule, and then modifying the reactive site derived from the activity-based labeling molecule with a reporter enzyme-labeled molecule. When a conjugation molecule is involved, the modification step involves labeling the labeling site derived from the activity-based labeling molecule that labels the target protein with a conjugation molecule in one or multiple steps, and then modifying the end of the conjugation molecule with a reporter enzyme-labeled molecule.

[0100] Contact between the target protein or its labeled form and the conjugate molecule or reporter enzyme-labeled molecule can be performed in the same manner as described for the contact in the labeling step, and therefore further explanation is omitted. For example, by adding an excess of the conjugate molecule or reporter enzyme-labeled molecule to the target protein or its labeled form, the activity / reactivity of the active / reactive site can be evaluated while suppressing the influence of labeling efficiency. Alternatively, for example, after contact with the respective conjugate molecule or reporter enzyme-labeled molecule, purification by ultrafiltration may be performed.

[0101] The labeling step according to one embodiment may include contacting a protein of interest modified with a reporter enzyme with the protein of interest so that one molecule of the protein of interest binds to each complex of a bead and an antibody or antigen-binding fragment thereof that binds to the protein of interest. Such contact may be carried out, for example, in a solution containing 0.0002 to 0.5, 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05 number-average molecules of the protein of interest per bead. In this embodiment, the protein of interest modified with the reporter enzyme is captured by the bead through an antigen-antibody reaction. Therefore, due to the specificity of the antibody or antigen-binding fragment thereof that binds to the protein of interest, evaluation can be performed under conditions in which a solution containing one molecule of the protein of interest is a solution that selectively contains the protein of interest (a solution containing only one molecule of the target protein as a biopolymer). In this case, only components containing the target protein as a constituent element are captured on the beads, so the target protein modified with the reporter enzyme can be separated and captured even if the target protein modified with the reporter enzyme is not separated from the reporter enzyme or the conjugated molecule in the modification step.

[0102] The antibody that binds to the target protein may be any antibody that selectively binds to the target protein, and may be, for example, a monoclonal antibody or a polyclonal antibody, with a monoclonal antibody being preferred from the viewpoint of the stability of the evaluation results. Such a monoclonal antibody may be, for example, a commercially available monoclonal antibody, or may be, for example, a monoclonal antibody obtained by a method commonly performed by those skilled in the art using a hybridoma prepared from antibody-producing cells collected from an animal immunized with the target protein. The antigen-binding fragment may be any antibody that selectively binds to the target protein, and may be, for example, Fab, F(ab') ２ , scFv or VHH.

[0103] The antibody or its antigen-binding fragment can be modified onto beads according to methods commonly used by those skilled in the art. For example, the antibody or its antigen-binding fragment may be modified onto the beads by physical adsorption by adding the beads to an aqueous solution in which the antibody or its antigen-binding fragment has been dissolved. Alternatively, the antibody or its antigen-binding fragment may be modified onto the beads by forming a covalent bond between a functional group on the surface of the beads and a functional group on the antibody. Examples of methods for covalently modifying beads with an antibody or an antigen-binding fragment thereof include: (1) a method in which beads having a carboxyl group on their surface are used, and the carboxyl group is activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to form an amide bond with an amino group on the antibody; (2) a method in which formyl groups are first introduced into an antibody or an antigen-binding fragment thereof by contacting the antibody with glutaraldehyde, and then, using beads having an amino group on their surface, the amino groups on the bead surface are reductively bonded to the formyl groups on the antibody; and (3) a method in which maleimide groups are first introduced onto the bead surface, and then the maleimide groups are reacted with mercapto groups on the antibody or antigen-binding fragment thereof to form a thioether bond.

[0104] In one embodiment, the beads used may be fluorescent. In this case, the presence or absence of beads can be evaluated based on fluorescence. As a result, for example, a solution in which fluorescence from beads is detected can be selected as the solution to be evaluated. Furthermore, for example, according to this embodiment, the solution to be evaluated can be determined without using two or more antibodies that bind to one enzyme, as in the digital ELISA method. This can reduce the risk of reduced evaluation accuracy due to, for example, antibody competition.

[0105] In one embodiment, the labeling step may further include contacting the target protein with an antibody or an antigen-binding fragment thereof that is capable of binding to the target protein and that has been modified with a reporter enzyme. In this case, the detection step described below may also measure the enzymatic reaction of the reporter enzyme that modifies the antibody or its antigen-binding fragment in a solution containing one molecule of the protein. In this embodiment, as in the case where the beads are fluorescent, the target protein in solution can be detected using the enzymatic reaction of the reporter enzyme, with the signal derived from the reaction product generated by the reporter enzyme metabolizing the substrate as an indicator. The reporter enzyme and its substrate can be the same as those described for labeling the active / reactive site. However, in this case, it is preferable to use reporter enzymes that modify the active / reactive site and reporter enzymes that modify the antibody or its antigen-binding fragment, which are orthogonal to each other in their enzymatic reactions. In this case, it is preferable to use substrates for both enzymes that produce reaction product signals that can be distinguished from each other by wavelength, etc. In this case, the antibody or antigen-binding fragment thereof may be the same as that described for the antibody or antigen-binding fragment thereof used to modify the beads. In addition, modification of the antibody or antigen-binding fragment thereof with a reporter enzyme may be performed in the same manner as that described for the method of binding a binding site or linker site to an enzyme in a reporter enzyme-labeled molecule that labels an activity-based labeling molecule.

[0106] <Preparation Step> In the preparation step, a solution containing one molecule of a target protein modified with a reporter enzyme and a substrate for the reporter enzyme is prepared. In the example shown here, the target protein modified with a reporter enzyme is prepared in the modification step. In the preparation step, for example, a mixture containing one molecule of the target protein modified with a reporter enzyme may first be prepared, and then the substrate for the reporter enzyme may be added to the mixture. Alternatively, for example, a mixture (pre-encapsulation mixture) containing multiple target proteins modified with a reporter enzyme and the substrate for the reporter enzyme may first be prepared, and then the pre-encapsulation mixture may be divided into solutions each containing a single molecule of the target protein modified with a reporter enzyme. From the viewpoint of simplicity, the latter is preferred. Thus, in a preferred embodiment, the preparation step may include encapsulating the pre-encapsulation mixture containing the target protein modified with a reporter enzyme and the substrate for the reporter enzyme in a space closed to the mixture so that each mixture contains a single molecule of the target protein.

[0107] For example, if the target protein modified with a reporter enzyme is not captured on beads, the preparation step may involve dividing the solution so that one molecule of the target protein is stochastically contained. Alternatively, for example, if the target protein modified with a reporter enzyme is captured on beads, the preparation step may involve placing the beads in a microwell (placement step) so that one bead capturing the target protein modified with a reporter enzyme is contained. In the latter case, the protein bound to the antibody or its antigen-binding fragment is isolated from the reaction solution used in the labeling step by placing the beads, so that the activity / reactivity of the active / reactive site can be evaluated under conditions in which the protein is substantially concentrated. Therefore, when a protein in a biological sample (such as a specimen) is the target of evaluation, the activity / reactivity of the active / reactive site can be evaluated even using a sample with a target protein concentration several orders of magnitude lower than in conventional evaluation methods using a single-molecule evaluation system prepared by the stochastic diffusion of biopolymers.

[0108] When the preparation step includes encapsulating the pre-encapsulation mixed solutions in a space closed to the mixed solutions so that each mixed solution contains one molecule of the target protein, the number-average number of molecules of the target protein contained in one solution may be, for example, 0.0002 to 0.5, or may be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05. When the preparation step includes a placement step, the number-average number of beads contained per microwell may be, for example, 0.0002 to 0.5, or may be 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05. When the number-average number of molecules of the target protein per solution or the number-average number of beads per microwell is within the above range, the number of molecules of the target protein in the mixed solution in which the target protein has been encapsulated is likely to be one molecule, making it easier to prevent a signal from being measured in detection from a mixed solution in which two or more molecules of the target protein have been encapsulated. The number-average number of molecules of the target protein contained in each of the above-mentioned mixed solutions may be, for example, the value obtained by multiplying the concentration of the target protein in the pre-encapsulation mixed solution [molecules / L] by the volume of the aqueous solution in the preparation step (e.g., the volume of the well) [L]. The number-average number of beads contained in each of the above-mentioned mixed solutions may be, for example, the value obtained by multiplying the concentration of the target protein in the pre-encapsulation mixed solution [molecules / L] by the volume of the aqueous solution in the preparation step (e.g., the volume of the well) [L].

[0109] The concentration of the reporter enzyme substrate in the pre-encapsulation mixed solution is not particularly limited as long as it is a concentration at which a signal can be detected in the detection step, and may be, for example, 1 μM to 1000 μM, 3 μM to 700 μM, 10 μM to 500 μM, or 30 μM to 300 μM. The concentration of the reporter enzyme substrate in the pre-encapsulation mixed solution is usually the same as the concentration of the reporter enzyme substrate after the target protein in each mixed solution is encapsulated in a space closed relative to the mixed solution so that it constitutes a single molecule.

[0110] The pH of the aqueous solution prepared in the preparation step is not limited as long as it allows an enzymatic reaction by the reporter enzyme to occur, and may be, for example, within the range of the optimal pH of the reporter enzyme ±3.0, the range of the optimal pH of the reporter enzyme ±2.0, the range of the optimal pH of the reporter enzyme ±1.5, or the range of the optimal pH of the reporter enzyme ±1.0. Specifically, the pH of the aqueous solution prepared in the preparation step may be, for example, 3.5 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 6.0 or more, 6.5 or more, 7.0 or more, 7.5 or more, 8.0 or more, 8.5 or more, or 9.0 or more, or may be 9.5 or less, 9.0 or less, 8.5 or less, 8.0 or less, 7.5 or less, 7.0 or less, 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less, 4.5 or less, or 4.0 or less.

[0111] The mixture prepared in the preparation step may contain other components in addition to the reporter enzyme-modified target protein or beads capturing the protein and the reporter enzyme substrate. Examples of other components include buffers (e.g., phosphate buffer, HEPES buffer, and Tris buffer), pH adjusters (e.g., hydrochloric acid, citric acid, sodium hydroxide, sodium carbonate, and sodium bicarbonate), ion concentration adjusters (e.g., calcium chloride and magnesium chloride), osmotic pressure adjusters (e.g., sodium chloride), antioxidants (e.g., dithiothreitol (DTT)), and surfactants (e.g., Triton (registered trademark) X-100, CHAPS, and Tween (registered trademark) 20).

[0112] In the evaluation method according to the present disclosure, when the evaluation system is an oil-sealed chamber system, the placement step may include, in order, pouring an aqueous solution containing a reporter enzyme-modified target protein or beads capturing the protein into multiple storage compartments of a microchamber array having a hydrophobic surface, and a reporter enzyme substrate into each of the multiple storage compartments through the openings so that the number-average number of beads filled in each of the multiple storage compartments is 0.0002 to 0.5, and then blocking the openings with a hydrophobic solvent. Examples of such microchamber arrays include those described in Patent Document 2 and Non-Patent Document 4, and more specifically, a Simoa® disk (Quanterx) can be used. The volume of the solution in each of the multiple storage compartments may be, for example, 1 fL to 500 fL, 5 fL to 100 fL, 10 fL to 75 fL, or 20 fL to 60 fL. The number-average molecular number of the peptidase filled in each of the plurality of storage sections may be, for example, 0.0002 to 0.5, 0.0005 to 0.1, 0.001 to 0.07, or 0.003 to 0.05.

[0113] In the evaluation method according to the present disclosure, when the evaluation system is an oil-sealed chamber system, a substrate for the reporter enzyme that is highly soluble in water and its reaction product can be preferably used in order to prevent leakage into the hydrophobic solvent. For example, a compound that distributes 80% or more of the compound in the PBS layer when 1 μM of the compound is added to a two-layer system of octanol and phosphate-buffered saline (PBS) at pH 7.4 in a volume ratio of 1:1 can be used as the reporter enzyme substrate.

[0114] In the evaluation method according to the present disclosure, when the evaluation system is an oil seal chamber system, from the viewpoint of making it easier to observe fluorescence, a compound having a product of the molar extinction coefficient [L / mol cm] at the maximum absorption wavelength of the reaction product and the fluorescence quantum yield of 10,000 or more can be preferably used as the substrate for the reporter enzyme. From the same viewpoint, a compound having a maximum fluorescence wavelength of 450 nm or more can be preferably used.

[0115] <Detection Step> In the detection step, the activity-based labeling molecule that labels the target protein is detected in the solution obtained in the preparation step using the enzymatic reaction of the reporter enzyme. As described above, the reporter enzyme is modified at the labeling site derived from the activity-based labeling molecule using a high-affinity pair and is carried out so that the modification proceeds sufficiently. Furthermore, from a solution containing one molecule of target protein modified with the reporter enzyme, the enzymatic reaction of the reporter enzyme produces a reaction product that allows a detectable signal. Therefore, although not particularly limited, in the detection step, each molecule of the target protein can be detected by binarizing whether it has been labeled with the activity-based labeling molecule or not. The threshold value of the signal intensity when detecting by binarization can be appropriately set by those skilled in the art as a value that can be distinguished from background signals.

[0116] In the detection step, when light irradiation is required to detect the signal of the reaction product, the reaction product is irradiated with light of a wavelength corresponding to the reaction product. For example, when the signal is fluorescence, light of a wavelength corresponding to the excitation wavelength of the reaction product may be irradiated. Furthermore, when the signal is Raman scattered light, light of a wavelength at which Raman scattering occurs in the reaction product may be irradiated.

[0117] For example, in the detection step, the proportion of proteins labeled with the activity-based labeling molecule among the tested target proteins (i.e., the probability of labeling) may be detected based on the presence or absence of labeling of the target proteins in each solution with the activity-based labeling molecule. In this case, for example, a target protein with a high percentage of labeling may be determined to have high activity / reactivity of the active / reactive site, and a target protein with a low percentage of labeling may be determined to have low activity / reactivity of the active / reactive site. For example, a target protein with a high percentage of labeling may be determined to have a high percentage of active / reactive sites. The degree of labeling may be determined, for example, based on the presence or absence of a significant difference in a comparison between two or more groups. For example, a target protein in which labeling has occurred may be determined to have activity / reactivity of the active / reactive site, and a target protein in which labeling has not occurred may be determined to not have activity / reactivity of the active / reactive site. The presence or absence of labeling may be determined for each solution, or based on the presence or absence of a significant difference in a comparison with a solution (negative control group) to which the activity-based labeling molecule and / or the reagents necessary for its labeling have not been administered.

[0118] Furthermore, for example, if one molecule of a target protein has multiple active / reactive sites and is therefore labeled with multiple activity-based labeling molecules, the intensity of the signal detected from each solution in the detection step will be proportional to the number of activity-based labeling molecules that have labeled one molecule of the target protein. Thus, for example, in the detection step, the number of active / reactive sites present in the target protein may be evaluated using the signal intensity detected in each solution as an index.

[0119] <Action and Effect> As described above, the method for evaluating the activity / reactivity of an active / reactive site of a protein according to the first embodiment comprises a labeling step of labeling a target protein with an activity-based labeling molecule, and a detection step of detecting, in a solution containing one molecule of the labeled target protein, the activity-based labeling molecule that has labeled the target protein using an enzymatic reaction of a reporter enzyme (first reporter enzyme) that modifies the activity-based labeling molecule, wherein the activity-based labeling molecule reacts with the active / reactive site of the target protein and forms a covalent bond with the target protein or its active / reactive site after the reaction, and the labeling step is an evaluation method comprising contacting the activity-based labeling molecule with the target protein.

[0120] According to the evaluation method of this embodiment, like an ELISA method such as a digital ELISA method, an enzymatic reaction of a reporter enzyme (first reporter enzyme) is used, and the activity / reactivity of an active / reactive site of a protein can be evaluated at the single molecule level using, as an indicator, a signal derived from a reaction product generated when a substrate is metabolized by the reporter enzyme (first reporter enzyme).

[0121] Furthermore, according to the evaluation method of this embodiment, even if the activity-based labeling molecule itself is not a molecule capable of detecting a signal, the labeling of a protein by the activity-based labeling molecule can be detected using the enzymatic reaction of the reporter enzyme (first reporter enzyme). This also allows the activity / reactivity of the protein's active / reactive site to be evaluated at the single-molecule level. This allows the activity / reactivity of the protein's active / reactive site to be evaluated at the single-molecule level without the need to newly develop a dedicated activity-based labeling molecule (e.g., a molecule whose signal increases upon reaction with the protein) with a detectable signal for each target protein. Furthermore, since it is possible to simultaneously select an activity-based labeling molecule that is preferable in terms of reactivity with the protein (e.g., reaction rate, selectivity), and a reporter enzyme (first reporter enzyme) that is preferable in terms of signal strength, the activity / reactivity of the protein's active / reactive site can be favorably evaluated at the single-molecule level.

[0122] In one aspect of the evaluation method according to the first embodiment, the activity-based labeling molecule may include a reactive site that reacts with the active / reactive site of the target protein and a labeling site (first labeling site), and the labeling step may further include contacting the target protein with the activity-based labeling molecule and then directly or indirectly modifying the labeling site (first labeling site) derived from the activity-based labeling molecule with a reporter enzyme (first reporter enzyme). In another aspect of the evaluation method according to this embodiment, in which the labeling site is directly modified with a reporter enzyme, the labeling step may include contacting the target protein with the activity-based labeling molecule and then further contacting the target protein with a reporter enzyme-labeled molecule (first reporter enzyme-labeled molecule), and the reporter enzyme-labeled molecule (first reporter enzyme-labeled molecule) may be a molecule that includes a binding site (first binding site) that specifically binds to the labeling site (first labeling site) of the activity-based labeling molecule and a reporter enzyme (first reporter enzyme) modified thereto.

[0123] In one aspect of the evaluation method according to the first embodiment, the labeling step may further include contacting the target protein with an antibody or an antigen-binding fragment thereof that is capable of binding to the target protein and that is modified with a reporter enzyme (third reporter enzyme), and may include measuring the enzymatic reaction of the reporter enzyme (third reporter enzyme) in a solution containing one molecule of the labeled target protein. According to this aspect, the protein in the solution can be detected using the enzymatic reaction of the reporter enzyme (third reporter enzyme) and using as an indicator a signal derived from a reaction product generated when the reporter enzyme (third reporter enzyme) metabolizes a substrate. This allows the enzymatic reaction of the reporter enzyme (third reporter enzyme) to confirm that one molecule of the target protein is present in the solution. For example, if proteins are arranged so that one molecule of the target protein can be stochastically contained in the solution, a solution containing one molecule of the target protein can be selected from candidate solutions using the enzymatic reaction of the reporter enzyme (third reporter enzyme).

[0124] <Post-translational Modification Evaluation Method> A second embodiment of the present disclosure is a method for evaluating post-translational modifications of proteins. An outline of the evaluation method of this embodiment is shown in FIG. 2. In the evaluation method of this embodiment, a target protein is first labeled with a post-translational modification labeling molecule. Then, the post-translational modification labeling molecule, which has been further modified with a reporter enzyme, is detected at the single-molecule level based on the activity of the reporter enzyme. As such, the evaluation method of the second embodiment is similar to the evaluation method of the first embodiment, except that a post-translational modification labeling molecule is used instead of an activity-based labeling molecule. That is, the evaluation method of the second embodiment can be carried out in the same manner as the evaluation method of the first embodiment and the detailed evaluation examples described therein, except that a post-translational modification labeling molecule is used instead of an activity-based labeling molecule. Therefore, hereinafter, only the post-translational modification and the post-translational modification labeling molecule, which are differences from the first embodiment, will be described, and other explanations will be omitted.

[0125] The evaluation method of the second embodiment can also be carried out simultaneously with the evaluation method of the first embodiment. In this case, both the activity / reactivity of the active / reactive site and post-translational modification can be evaluated for a single target protein molecule. This makes it possible to evaluate, for example, the effect of post-translational modification on the activity / reactivity of the active / reactive site of a protein.

[0126] <Post-translational Modification> The post-translational modification according to the present disclosure is a modification that occurs after a protein is biosynthesized. The post-translational modification is not particularly limited, and may be, for example, the addition of an atom, a functional group, or an oligomer, or oxidation or reduction, and in one aspect, may be phosphorylation, glycosylation, hypersulfur modification, or disulfide bond. In the evaluation method of this embodiment, a site in a target protein that has been post-translationally modified (post-translation modification site) is labeled with a post-translational modification labeling molecule.

[0127] <Post-translational modification labeled molecule> The post-translational modification labeled molecule is a molecule that selectively binds to a post-translational modification site of the protein. The post-translational modification labeled molecule may have an interaction site that interacts with the post-translational modification site and a labeling site, and may further have a linker site. The labeling site and linker site in the post-translational modification labeled molecule and the relationship between each site are the same as those described for the labeling site, linker site, and reaction site of the activity-based labeled molecule in the first embodiment. Therefore, only the interaction site, which is the difference between the post-translational modification labeled molecule and the activity-based labeled molecule, will be described, and a description of the other sites and the relationship between each site will be omitted.

[0128] In one embodiment, the post-translationally modified labeling molecule may be a molecule with a molecular weight (g / Mol) of 5,000 or less, or may be a molecule with a molecular weight of 2,500 or less, or 1,000. In this case, the volume occupied by the post-translationally modified labeling molecule in the space surrounding the protein is smaller than when, for example, an antibody is used as the interaction site. This can suppress inhibition of the reaction between the protein and the activity-based labeling molecule. Furthermore, when an antibody is further bound to the protein, competition between the labeling molecule and the antibody (e.g., competition due to multiple antibodies) can be suppressed.

[0129] <Interaction Site> The interaction site is a site that interacts with and binds to a post-translational modification site, and the bond may be a covalent or non-covalent bond. The interaction site may be any site that interacts with a post-translational modification site, and can be appropriately selected by those skilled in the art according to the structure of the post-translational modification site. For example, the interaction site may be an antibody or antigen-binding fragment specific to a target protein having a post-translational modification site. Such an antibody or antigen-binding fragment may be a protein that binds to a target protein that has been post-translationally modified, but not to a target protein that has not been post-translationally modified. Such an antibody or antigen-binding fragment can be prepared by producing an antibody or antigen-binding fragment that binds to a target protein that has been post-translationally modified according to a conventional method, and then negatively selecting, by affinity chromatography or the like, from among the antibodies or antigen-binding fragments that bind to a target protein that has not been post-translationally modified. When the post-translational modification is phosphorylation, the interaction site may be, in addition to an antibody, for example, a Phos-tag (Eiji Kinoshita et al., "Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex", Dalton Trans., 2004, 1189-1193). Phos-tag forms a strong coordinate bond with phospho and can therefore be used as an interaction site for phospho at the post-translational modification site. When the post-translational modification is glycosylation, the interaction site may be, in addition to an antibody, for example, a lectin or aminooxy (-O-NH ２) may be used. Those skilled in the art can appropriately select a lectin that specifically binds to the glycosylation target to be evaluated. For example, lectins registered in databases such as the RCSB Protein Data Bank (RCSB PDB) can be used. Aminooxy can form a covalent bond with sialic acid, a site added to proteins by post-translational modification (Masaki Kurogochi et al., "Reverse Glycoblotting Allows Rapid-Enrichment Glycoproteomics of Biopharmaceuticals and Disease-Related Biomarkers", Angew. Chem. Int. Ed. 46 (2007), 46, 8808-8813).

[0130] <Actions and Effects> As described above, the evaluation method of the second embodiment includes a labeling step of labeling a target protein with a post-translational modification labeling molecule, and a detection step of detecting, in a solution containing one molecule of the labeled target protein, the post-translational modification labeling molecule that has labeled the target protein, using an enzymatic reaction of a reporter enzyme (second reporter enzyme) that modifies the post-translational modification labeling molecule, wherein the labeling step includes contacting the target protein with the post-translational modification labeling molecule, and the detection step includes detecting, in a solution containing one molecule of the labeled target protein, the post-translational modification labeling molecule that has labeled the target protein, using an enzymatic reaction of a reporter enzyme (second reporter enzyme) that modifies the post-translational modification labeling molecule, wherein the post-translational modification labeling molecule is a molecule that selectively binds to a post-translational modification site in the target protein.

[0131] According to the evaluation method of this embodiment, like ELISA methods such as digital ELISA, the presence or absence of post-translational modification can be evaluated in a single-molecule system using an enzymatic reaction of a reporter enzyme (second reporter enzyme) and a signal derived from a reaction product generated by the reporter enzyme (second reporter enzyme) metabolizing a substrate as an indicator. Furthermore, by simultaneously performing the evaluation method of the first embodiment, both the activity / reactivity of an active / reactive site and post-translational modification can be evaluated in a single molecule of a target protein using the enzymatic reaction of the corresponding reporter enzymes (first reporter enzyme, second reporter enzyme) as indicators. This allows, for example, the effect of post-translational modification on the activity / reactivity of an active / reactive site of a protein to be evaluated at the single protein molecule level.

[0132] Furthermore, in one aspect of the evaluation method according to the second embodiment, the post-translationally modified labeled molecule may include an interaction site that interacts with the post-translation modification site and a labeling site (second labeling site), and the labeling step may further include contacting the target protein with the post-translationally modified labeled molecule and then directly or indirectly modifying the labeling site (second labeling site) derived from the post-translationally modified labeled molecule with a reporter enzyme (second reporter enzyme). Furthermore, in an aspect of the evaluation method according to this aspect in which the labeling site is directly modified with a reporter enzyme, the labeling step may include contacting the target protein with the post-translationally modified labeled molecule and then further contacting the protein with a reporter enzyme-labeled molecule (second reporter enzyme-labeled molecule), and the reporter enzyme-labeled molecule (second reporter enzyme-labeled molecule) may be a molecule including a binding site (second binding site) that specifically binds to the labeling site (second labeling site) of the post-translationally modified labeled molecule and a reporter enzyme (second reporter enzyme) modified thereto.

[0133] <Applications> The evaluation method according to the present disclosure can be used for research purposes such as drug discovery research and biological research. For example, a substance evaluated by the evaluation method according to the present disclosure as having changed the activity / reactivity and / or post-translational modification of a target protein in a desired direction for disease treatment / prevention can be selected as an active ingredient of a pharmaceutical for treating and / or preventing a disease in which the target protein is involved. Furthermore, for example, based on the determination by the evaluation method according to the present disclosure that the activity / reactivity and / or post-translational modification of a target protein has changed in a certain environment, it can be evaluated whether the activity / reactivity and / or post-translational modification of the target protein is responsive to the environment. Furthermore, for example, by evaluating the activity / reactivity and post-translational modification of the target protein at the single molecule level, the effect of post-translational modification of the target protein on activity / reactivity can be evaluated.

[0134] The evaluation method according to the present disclosure can be used for the purpose of, for example, diagnosis, diagnostic assistance, or obtaining data for diagnosis. For example, when the proportion of active / reactive sites and / or post-translational modifications in a target protein differs between a patient with a certain disease and a healthy subject, an indicator of whether the subject is affected by the disease can be provided based on the difference in the proportions.

[0135] The present disclosure will be described in more detail below with reference to examples, but the present disclosure should not be construed as being limited to the following examples. In these examples, error bars of results obtained from multiple samples are shown as mean ± standard deviation for n=3, and statistical tests were performed by Student's T-test.

[0136] Example 1: Labeling of Protein Reactive Sites Lysine residues, which are reactive sites in proteins, were labeled. Prostate-specific antigen (PSA) was used as a model protein to be detected. NHS ester was used as the reactive site that reacts with lysine residues in the activity-based labeling molecule. Alkaline phosphatase (ALP) was used as the reporter enzyme to detect the activity-based labeling molecule. In the following examples, PSA and ALP were used after the commercially available recombinant protein buffer was exchanged with a phosphate buffer solution at pH 7.4 using an Amicon filter (50 kDa, Merck, UFC505096). sTG-mPhos (Non-Patent Document 3) was used as a fluorescent probe to detect ALP activity. This fluorescent probe is weakly or non-fluorescent before the enzymatic reaction, but the fluorescent dye produced after metabolism by the enzyme becomes highly fluorescent. Therefore, the fluorescence intensity of the fluorescent dye produced by metabolism of this fluorescent probe can be used as an indicator of the amount of reporter enzyme present.

[0137] Step 1: Labeling the reactive site of the target protein; Preparation of azide-modified PSA (target protein). 250 μL of Calibrator H (containing 76.3 pg / mL of PSA) included in the Simoa® Homebrew Assay Starter Kit (Quanterix, 101351) for calibration was purified using an Amicon filter (50 KDa, Merck, UFC505096). The solution that did not pass through the filter was diluted with pH 7.4 phosphate buffer (hereinafter simply referred to as "PBS") to obtain a PSA solution with a total protein concentration of 2.7 μM (1 molar equivalent), as measured using a NanoDrop® spectrophotometer (Thermo Scientific). This PSA solution also contained proteins other than PSA derived from Calibrator H. Azide-PEG4-NHS (FUJIFILM Wako, AG003T2B) was added to a final concentration of 81 μM (30 molar equivalents). The reaction solution was allowed to react at room temperature for 30 minutes and then purified with an Amicon filter (50 kDa, Merck, UFC505096). The solution that did not pass through the filter was used as an azide-modified PSA (PSA-Azide) solution. Note that step 1 was performed only under Linker (+) conditions (Figure 3). Under Linker (-) conditions (Figure 3), a solution of PSA at the same concentration dissolved in PBS was used in place of the PSA-Azide solution in the subsequent step 3.

[0138] [Step 2: Preparation of alkyne-modified ALP (reporter enzyme-labeled molecule)] ALP (Roche, 3359123001) was dissolved in PBS to a final concentration of 62.6 μM (1 molar equivalent). Alkyne-PEG5-NHS (Tokyo Chemical Industry, P2283) was added to the solution to a final concentration of 1879 μM (30 molar equivalents). The reaction solution was reacted at room temperature for 30 minutes and then purified with an Amicon filter (50 kDa). As a result, an alkyne-modified ALP (ALP-Alkyne) solution was obtained as the solution that did not pass through the filter.

[0139] [Step 3: Labeling of PSA (target protein) with ALP (reporter enzyme)] The PSA-azide solution obtained in step 1 and the ALP-alkyne solution obtained in step 2 were mixed so that the final concentration of PSA-azide was 0.25 μM and the final concentration of ALP-alkyne was 2.5 μM (concentration ratio 1:10). ４ 1 mM PSA, 1 mM ascorbic acid, and 3 mM BTTP (Vector Laboratories, CCT-1414, CAS No. 1334179-85-9) were added, and the click reaction was carried out overnight at room temperature. This resulted in a solution containing ALP-labeled PSA (PSA-ALP). Note that step 3 was carried out only under Click(+) conditions (Figure 3). Under Click(-) conditions (Figure 3), the PSA-Azide solution obtained in step 1 and the ALP-Alkyne solution obtained in step 2 were mixed at a volume ratio of 1:10, and this solution was used in the subsequent step 4.

[0140] [Step 4: Capture of PSA-ALP by beads] This step was carried out in accordance with the Quanterix manual and the published online protocol "SiMOA total Rab10 Homebrew Assay" (dx.doi.org / 10.17504 / protocols.io.dm6gp3188vzp / v1). Detailed procedures are described below. 488-dyed singleplex beads (Quanterix, 104007) were suspended in Bead Conjugation Buffer (Quanterix, 100453), and EDC was added to a concentration of 0.3 mg / ml. The beads were incubated at 4 ° C for 30 minutes to activate the carboxylates on their surface. An anti-PSA antibody (Homebrew PSA Capture, Quanterix, 100463) was added to the mixture at a concentration of 0.2 mg / ml, and the mixture was allowed to react at 4°C for two hours. The resulting antibody-modified beads were then incubated at room temperature for 45 minutes in PBS containing BSA for blocking. This resulted in beads whose surfaces were labeled with anti-PSA antibodies (anti-PSA antibody-labeled beads). The solution containing PSA-ALP obtained in step 3 was diluted 10-fold with HEPES, and 100 μL of the diluted solution was loaded onto a 96-well microplate (Thermo Fisher Scientific, 249944). The anti-PSA antibody-labeled beads were diluted to 1.4 × 10 ９ 25 μL of beads / mL was added (number of beads: 3.5 × 10 ７beads). The resulting mixture was reacted for 30 minutes while shaking at 800 rpm at 30°C to obtain beads capturing PSA-ALP (PSA-ALP beads). The resulting PSA-ALP beads were washed with buffer A (Quanterix Simoa wash buffer A), buffer B (Quanterix Simoa wash buffer B), and water using a microplate washer (BIOTEK, 405 TS). From the final measurement results of this example, the concentration of PSA-ALP in the mixture reacted with the anti-PSA antibody-labeled beads in this step was estimated to be on the order of several tens of pg / mL in terms of PSA. Therefore, it was inferred that the number of PSA-ALP particles in the mixture was smaller than the number of beads.

[0141] [Step 5: Detection of reporter enzyme activity using a microdevice] Using an SR-X (Quanterix), the beads obtained in step 4 were suspended in HEPES-Na buffer (pH 7.4) containing 0.25 mM Triton-X-100. sTG-Phos (final concentration 30 μM) was added to the suspension. 40 μL of the resulting mixture was introduced into a Simoa® disk (Quanterix), a microchamber array. Next, 80 μL of Fluorinert® Simoa sealing oil (Quanterix, 102767) was introduced into the microchamber array as a hydrophobic solvent, and the aqueous solution not introduced into the well was removed, forming a W / O droplet in the well. This introduction stochastically prepared wells containing a single bead. After incubating the microchamber array at room temperature for 20 hours, fluorescent images were acquired using an epi-illumination microscope Ti2 (Nikon) equipped with a 20x objective lens (Plan Apo 20x), an sCMOS camera (ORCA-Fusion C14440, Hamamatsu Photonics), a white light unit (X-Cite Xylis, Optoscience), and a motorized stage.

[0142] [Results] Figure 3 shows the results of observing the fluorescence of the fluorescent dye generated by the metabolism of reporter enzyme in an oil-sealed chamber system for beads capturing PSA whose reactive lysine residues are modified with ALP in Example 1. According to Figure 3, under the conditions of Linker (+) and Click (+), that is, under the conditions that the reactive lysine residues of PSA are modified with ALP, the fluorescent signal derived from the enzyme activity of ALP (i.e., the fluorescence derived from the metabolic product of ALP) was observed. From this result, it was shown that the reactivity of the lysine residues of PSA can be detected by covalently labeling the lysine residues, using the metabolic reaction of the reporter enzyme as an indicator.

[0143] Example 2: Labeling of the active site of a purified enzyme 1 Labeling of the active site of an enzyme was performed. PSA was used as a model protein to be detected. Since PSA is also known as kallikrein 3 (KLK3), hereinafter PSA may also be referred to as KLK3. PSA is a type of serine hydrolase. PSA is an enzyme that is activated by being cleaved from proenzyme by thermolysin. Therefore, we investigated whether the presence or absence of activation by thermolysin results in a difference in the labeling efficiency of the active site. In the activity-based labeling molecule, a fluorophosphonate was used as the reactive site that reacts with the active site of PSA. The serine in the active center of an active serine hydrolase has strong nucleophilicity and therefore undergoes nucleophilic attack on the fluorophosphonate, resulting in selective and covalent labeling. The activity-based labeled molecule used was a molecule in which fluorophosphonate and biotin were linked via a linker (FP-biotin, Santa Cruz, sc-215056A, hereafter also referred to as "FP-biotin"). β-Galactosidase (β-Gal) was used as the reporter enzyme to detect the activity-based labeled molecule, and a molecule in which streptavidin and β-Gal were linked via a linker (SBG Concentrate Reagent, Quanterix, 103397) was used as the reporter enzyme-labeled molecule containing β-Gal. Resorufin β-D-galactopyranoside (Sigma-Aldrich, R4883-10MG) was used as the fluorescent probe to detect β-Gal activity. As the beads, fluorescent beads (Quanterix, 104007) were used.

[0144] [Preliminary Study: Determination of Labeling Conditions] Reaction conditions for labeling the active site of PSA were investigated using SDS-PAGE in combination with ABP, as has been done with conventional ABPP. Recombinant PSA (R&D Systems, 1344-SE-010) was dissolved in HEPES-Na buffer at pH 7.4 at 100 μg / ml to prepare a PSA solution. A 2 μg / ml thermolysin solution was prepared. Equal amounts of the PSA solution and thermolysin solution were mixed. The resulting solution was incubated at 37°C for 0, 1.5, 5, 15, or 45 minutes. After incubation, 1,10-phenanthroline solution was added to the solution at a concentration of 20 mM, followed by FP-biotin at a concentration of 20 μM. The resulting solution was incubated at room temperature overnight. The resulting solution was loaded so that the PSA concentration was 5 μg / mL and developed by SDS-PAGE at 500 V, 30 mA, and 60 minutes. The developed proteins were then transferred to a membrane at 500 V, 100 mA, and 60 minutes. The membrane was washed three times with Tris-Buffered Saline with Tween 20 (TBS-T), and then horseradish peroxidase (HRP)-labeled streptavidin (Streptavidin-HRP, Cell Signaling Technology, #3999) diluted 1 / 3000 with TBS-T was added to the membrane. After washing again three times with TBS-T, HRP was detected by chemiluminescence using the ECL Prime Western Blotting System, Cytiva, Cytiva RPN2232. In the chemiluminescence method, band intensity was quantified using Image J. Figure 4 shows the results of evaluating the labeling of the active site of PSA with ABP by SDS-PAGE and chemiluminescence in Example 2. Since high labeling efficiency was obtained when the activation time with thermolysin was 5 minutes, the activation time of PSA with thermolysin was set to 5 minutes.

[0145] [Step 1: Labeling the Active Site of the Purified Enzyme] Recombinant PSA (R&D Systems, 1344-SE-010) was dissolved in HEPES-Na buffer (hereinafter simply referred to as "HEPES") at pH 7.4 to prepare a PSA solution at 100 μg / ml. A 2 μg / ml thermolysin solution was prepared. Equal amounts of the PSA solution and the thermolysin solution were mixed. The resulting solution was incubated at 37°C for 5 minutes. After incubation, a 20 mM 1,10-phenanthroline solution was added to the solution, followed by 20 μM FP-biotin. The resulting solution was incubated overnight at room temperature. Note that under the conditions of PSA (untreated) (Figures 5 and 6), step 1 was not performed, and PSA that had not been subjected to step 1 was used in the subsequent step 2. In addition, under the PSA (inhibitor) conditions (FIG. 6), a molecule in which a fluorophosphonate and an azide are linked via a linker (ActivX (registered trademark) Azido-FP Serine Hydrolase Probe, manufactured by Thermo Scientific (registered trademark), 88316, hereinafter also referred to as "FP-azide") was added at a concentration of 20 μM, and the reaction solution was allowed to react at room temperature for 6 hours before the addition of FP-biotin.

[0146] [Step 2: Capture of PSA by beads] Fluorescent beads were modified with an anti-PSA antibody (Human / Monkey PSA Antibody Pair BSA and Azide-free Capture antibody, ab256313) according to the manufacturer's instructions to obtain fluorescent beads whose surfaces were labeled with the anti-PSA antibody (anti-PSA antibody-labeled beads). 100 μL of the PSA obtained in step 1 was loaded onto a microwell plate at a concentration of 600 pg / ml. 1.4 × 10 anti-PSA antibody-labeled beads were added to the beads. ９ Add 25 μL of beads / mL (number of beads: 3.5 × 10 ７The beads were then incubated at 30°C for 30 minutes with shaking at 800 rpm to obtain beads that captured PSA labeled with biotin at the active site (biotinylated PSA capture beads). The resulting biotinylated PSA capture beads were washed with buffer A, buffer B, and water using a microplate washer. Streptavidin-β-galactosidase conjugate was added to the beads at a final concentration of 100 pM and incubated at room temperature for 10 minutes. The beads in the resulting reaction solution were washed with buffer A, buffer B, and water using a microplate washer. The fluorescent beads thus obtained captured PSA whose serine residue at the active site was modified with β-Gal. Note that under conditions without PSA (Figure 5), step 2 was not performed, and beads that had not been subjected to step 2 were used in the subsequent step 3.

[0147] [Step 3: Detection of Enzyme Activity of Reporter Enzyme Using Microdevice] In the same manner as in step 5 of Example 1, the enzyme activity of the reporter enzyme was detected using a microdevice.

[0148] [Results] Figure 5 shows the results of observing the fluorescence of the fluorescent dye produced by the metabolism of the reporter enzyme in an oil-sealed chamber system for fluorescent beads capturing PSA in which the serine residues in the active site were modified with β-Gal in Example 2. Figure 6 shows the percentage (%) of wells in Example 2 in which red fluorescence derived from the metabolic products of β-Gal was observed among wells in which green fluorescence derived from the beads was observed. The results of Figures 5 and 6 show that a high percentage of wells in which red fluorescence derived from the metabolic products of β-Gal, the reporter enzyme corresponding to the labeling of the active site, were detected in PSA activated with thermolysin (PSA (thermolysin-treated for 5 min), activated PSA), whereas a low percentage was detected in PSA that had not been activated (PSA (untreated)). These results demonstrate that the activity of PSA at the active site can be detected using the metabolic reaction of the reporter enzyme as an indicator by covalently labeling the serine residue.

[0149] Example 3 Comparison of detection sensitivity in the presence or absence of reporter enzyme The detection sensitivity was compared between the case where the activity of a reporter enzyme that covalently modified a protein to be detected was detected based on the enzyme activity as in the method of the present disclosure, and the case where labeling with ABP as in the case of conventional ABPP was detected by chemiluminescence.

[0150] [Detection Based on Enzymatic Activity of Reporter Enzyme] PSA with biotinylated active sites (hereinafter also referred to as "biotinylated PSA") was prepared using recombinant PSA (R&D Systems, 1344-SE-010) in a manner similar to that used in step 1 of Example 2. Biotinylated PSA was reacted with fluorescent beads at concentrations of 0 pg / ml, 100 pg / ml, 200 pg / ml, 300 pg / ml, or 400 pg / ml for 30 minutes at 30°C while shaking at 800 rpm. By removing the liquid component from the reaction mixture, beads capturing PSA labeled with biotin at the active site (biotinylated PSA capture beads) were obtained as the solid component. Subsequently, the activity of PSA was evaluated based on the enzymatic activity of the reporter enzyme in the same manner as in step 2 (obtaining biotinylated PSA capture beads) and step 3 of Example 2.

[0151] [Detection by chemiluminescence] Biotinylated PSA was prepared using recombinant PSA (R&D Systems, 1344-SE-010) in the same manner as in step 1 of Example 2. Solutions of biotinylated PSA at concentrations of 0 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, and 100 ng / ml were prepared. Thereafter, the activity of PSA was evaluated by SDS-PAGE and chemiluminescence in the same manner as in the preliminary study in Example 2.

[0152] [Results] Figure 7 shows the results of evaluating PSA activity based on the enzymatic activity of the reporter enzyme in Example 3. In Figure 7, the horizontal axis shows the PSA protein concentration in pg (pg / mL) per mL, and the vertical axis shows the percentage (%) of wells in which red fluorescence from β-Gal metabolites was observed among wells in which green fluorescence from beads was observed. Figure 8 shows the results of evaluating PSA activity by detecting ABP labeling using chemiluminescence in Example 3. In the graph of Figure 8, the horizontal axis shows the PSA protein concentration in ng (ng / mL) per mL, and the vertical axis shows band intensity. In Figures 7 and 8, LOD indicates the detection limit determined by the 3σ method. Here, the detection limit determined by the 3σ method refers to the sample concentration estimated to give the result obtained by adding three times the standard deviation at 0 pg of the sample to the average value at 0 pg of the calibration curve. In Figures 7 and 8, LOQ indicates the limit of quantitation determined by the 10σ method. The limit of quantitation determined by the 10σ method refers to the sample concentration estimated to give the result of adding 10 times the standard deviation at 0 pg of sample to the average value at 0 pg of sample in the calibration curve. The results in Figures 7 and 8 revealed that when PSA activity was evaluated based on the enzyme activity of the reporter enzyme (LOQ: 4.36 pg / mL), the limit of quantitation was approximately 4000 times smaller than when evaluated by chemiluminescence (LOQ: 16.8 ng / mL), i.e., the sensitivity was approximately 4000 times higher. This result demonstrates that a method according to an embodiment of the present disclosure can detect the activity / reactivity of proteins at low concentrations. Therefore, it is believed that a method according to an embodiment of the present disclosure can also detect the activity / reactivity of proteins present in low amounts in vivo.

[0153] Example 4: Detection of enzyme activity in lysates Using lysates from two types of cell lines expressing PSA, we investigated whether it was possible to distinguish between the proportions of active PSA expressed in the two types of cell lines. The cell lines used were LNCap cells, an androgen-dependent cell line, and 22Rv1 cells, a non-androgen-dependent cell line. In addition, to confirm the validity of the measurement results, PSA activity was also measured in a bulk system.

[0154] [Lysate Preparation] The culture supernatant of adherently cultured LNCap cells or 22Rv1 cells was removed. Celllytic® M (Sigma-Aldrich, C2978-50ML) was added, and the cells were then detached and solubilized using a cell scraper (IWAKI, 9000-220). The lysate was then collected and centrifuged, and the supernatant was collected to obtain a cell lysate.

[0155] [Measurement of active PSA amount] Lysates of 22Rv1 cells and LNCap cells were prepared so that the protein concentration was 0.1 mg / ml. FP-biotin was added to the obtained cell lysate at a final concentration of 40 μM and incubated overnight at room temperature. The reaction solution was then diluted with Homebrew Detector Sample diluent (Quanterix, 101363) to a protein concentration of 800 ng / ml. PSA activity was evaluated based on the enzyme activity of the reporter enzyme using a method similar to steps 2 and 3 of Example 2. In the inhibitor group (Inhibitor group, Figure 10), FP-azide was added to the cell lysate before the protocol for the inhibitor-free group (Active group, Figure 10), and the solution was subjected to labeling with FP-biotin.

[0156] [Measurement of total PSA amount] Lysates of 22Rv1 cells and LNCap cells were diluted with Homebrew Detector Sample diluent (Quanterix, 101363) to a protein concentration of 800 ng / ml. 100 μL of each lysate was added to 1.4 × 10 PSA prepared in the same manner as in step 2 of Example 2. ９Fluorescent beads capturing PSA were obtained by reacting the beads with 25 μL of a suspension of anti-PSA antibody-labeled beads (beads / mL) at 30°C for 30 minutes while shaking at 800 rpm. The resulting fluorescent beads were washed with buffer A, buffer B, and water using a microplate washer. 250 ng / mL of biotinylated anti-PSA antibody was added and incubated at room temperature for 10 minutes. Streptavidin-β-galactosidase conjugate was added to a final concentration of 100 pM and incubated at room temperature for 10 minutes. The beads in the resulting reaction solution were washed with buffer A, buffer B, and water using a microplate washer. The total PSA content of the beads thus obtained was assessed based on the enzymatic activity of the reporter enzyme using the same method as in step 3 of Example 2.

[0157] [Control Experiment: Evaluation of PSA Activity by Bulk Assay] Cell lysate was diluted with HEPES to a protein concentration of 500 ng / ml. Suc-RPY-pNA (Cosmo Bio, AS-20586) was added to a final concentration of 4 mM, and the mixture was incubated at room temperature for 14 hours. The absorbance at 405 nm before and after incubation was measured using a microwell plate reader (Envison®, Perkin Elmer, 2013 Multilabel Reader). Suc-RPY-pNA is a dye whose absorbance at 405 nm increases when cleaved by active PSA.

[0158] [Results] Figure 9 shows the results of measuring the amounts of active PSA and total PSA in 22Rv1 cell and LNCap cell lysates in Example 4. Figure 10 shows the results of comparing the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites modifying active PSA was observed among wells in which green fluorescence derived from beads was observed, with different cell types and in the presence or absence of inhibitors in Example 4. Figure 11 shows the value obtained by dividing the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites modifying active PSA was observed among wells in which green fluorescence derived from beads was observed by the percentage (%) of wells in which red fluorescence derived from β-Gal metabolites modifying total PSA was observed among wells in which green fluorescence derived from beads was observed. Figure 12 shows the increase in absorbance before and after incubation of 22Rv1 cell and LNCap cell lysates in a bulk system for PSA activity measurement in Example 4. 9 to 11, the proportion of active PSA differed depending on the cell line, and the results showed the same tendency as the bulk assay in Fig. 12. These results demonstrate that the method according to one embodiment of the present disclosure can evaluate the activity / reactivity of proteins contained in cell lysates.

[0159] Example 5: Examination 1 of whether enzyme activity in plasma can be detected It is known that in the blood of healthy individuals, the proportion of active PSA is extremely low due to the action of antichymotrypsin, and the majority of PSA is inactive (Michael J Duffy, "Biomarkers for prostate cancer: prostate-specific antigen and beyond", Clin Chem Lab Med. 2020, 58(3):326-339). Therefore, whether PSA enzyme activity can be detected when plasma derived from healthy individuals is evaluated using a method according to an embodiment of the present disclosure was examined.

[0160] [Measurement of active PSA amount] FP-biotin was added to plasma derived from healthy subjects at a final concentration of 20 μM and incubated overnight at room temperature. The reaction solution was then diluted 300-fold with HEPES. Thereafter, PSA activity was evaluated based on the enzymatic activity of the reporter enzyme in the same manner as in Example 4.

[0161] [Measurement of Total PSA Amount] Plasma derived from a healthy subject was diluted 300-fold with HEPES. Thereafter, the total PSA amount was evaluated based on the enzyme activity of the reporter enzyme in the same manner as in Example 4.

[0162] [Results] Figure 13 shows the results of measuring the amount of active PSA and the amount of total PSA in plasma derived from healthy subjects in Example 5. As shown in Figure 13, many bright spots indicating the presence of PSA were observed when modified with the reporter enzyme via the anti-PSA antibody, whereas almost no bright spots indicating the presence of active PSA were observed when modified with the reporter enzyme via the activity-based labeling molecule. This result is consistent with the fact that "PSA contained in plasma derived from healthy subjects contains an extremely small proportion of active PSA, with the majority having no activity."

[0163] Example 6: Examination 2 of whether enzyme activity in plasma can be detected To further explore the suggestion obtained in Example 5, we examined whether there was a difference in the detected PSA activity when PSA was mixed with plasma from a healthy subject and then labeled with an activity-based labeling molecule, and when PSA was labeled with an activity-based labeling molecule and then mixed with plasma from a healthy subject.

[0164] [Labeling with activity-based labeling molecules after mixing with plasma from a healthy subject] 2 μL of an aqueous solution of PSA recombinant protein dissolved in HEPES at 120 ng / mL was mixed with 2 μL of plasma from a healthy subject. The mixture was incubated at room temperature for 3 hours. FP-biotin was added to the incubated mixture at a concentration of 20 μM. The resulting solution was incubated overnight at room temperature. The reaction solution was then diluted 150-fold with HEPES. Subsequently, PSA activity was evaluated based on the enzymatic activity of the reporter enzyme using the same method as in Example 4.

[0165] [Labeling with activity-based labeling molecules followed by mixing with plasma from healthy individuals] FP-biotin was added at 20 μM to 50 μL of an aqueous solution of PSA recombinant protein dissolved in HEPES at 10 μg / mL, and the mixture was incubated at room temperature for 6 hours. Labeled PSA was dissolved in HEPES to a concentration of 120 ng / mL, and 2 μL of this solution was mixed with 2 μL of plasma from a healthy individual. FP-biotin was added to the mixture at a concentration of 20 μM. The resulting solution was incubated overnight at room temperature. The reaction solution was then diluted 150-fold with HEPES. Subsequently, PSA activity was evaluated based on the enzymatic activity of the reporter enzyme using the same method as in Example 4.

[0166] [Results] Figure 14 shows the results of evaluating the activity of PSA when PSA was mixed with plasma derived from a healthy subject and then labeled with an activity-based labeling molecule (pre-mix group) and when PSA was labeled with an activity-based labeling molecule and then mixed with plasma derived from a healthy subject (post-mix group). As shown in Figure 14, even though the total amount of PSA was the same, strong PSA activity was detected when PSA was labeled with an activity-based labeling molecule before mixing with plasma derived from a healthy subject (post-mix group), whereas only weak PSA activity was detected when PSA was labeled with an activity-based labeling molecule after mixing with plasma derived from a healthy subject (pre-mix group). These results, consistent with the known fact that PSA loses activity in plasma, suggest the possibility that one embodiment of the present disclosure may enable the evaluation of the activity / reactivity of proteins contained in plasma.

[0167] Example 7: Labeling of the active site of a purified enzyme 2 Labeling of the active site of an enzyme was carried out under modification conditions for the protein to be detected and the reporter enzyme that were different from those in Example 2. Granzyme B was used as a model protein to be detected. In the activity-based labeling molecule, a fluorophosphonate was used as the reactive site that reacts with the active site of Granzyme B. Because Granzyme B is also a serine hydrolase, the serine in the active center is selectively and covalently labeled by the fluorophosphonate. FP-azide was used as the activity-based labeling molecule. β-Gal was used as the reporter enzyme to detect the activity-based labeling molecule. The modification with β-Gal was carried out by first labeling Granzyme B with FP-azide, followed by labeling the azide with a molecule in which alkyne and biotin are linked via a PEG4 linker (biotin-PEG4-Alkyne, manufactured by Tokyo Chemical Industry Co., Ltd., B6511, hereinafter also referred to as "alkyne-PEG4-biotin"), and then further labeling the biotin with the same streptavidin-β-galactosidase conjugate as in Example 2. The same resorufin β-D-galactopyranoside as in Example 2 was used as the fluorescent probe for detecting β-Gal activity. The same fluorescent beads as in Example 2 were used as the beads.

[0168] [Labeling of Granzyme B with Azide via Click Reaction] Granzyme B (R&D Systems, 2906-SE-0109) was dissolved at 100 μg / ml in HEPES-Na buffer at pH 7.4. FP-azide was added to the solution at a concentration of 20 μM, and the mixture was incubated overnight at room temperature. The resulting solution was diluted 10-fold with HEPES. Alkyne-PEG4-biotin was added to the solution at concentrations of 10 μM, CuSO4 at 1 mM, ascorbic acid at 1 mM, and BTTP at 3 mM, and the mixture was incubated at room temperature for 0 minutes, 30 minutes, 1 hour, or 2 hours. Through the above procedures, Granzyme B labeled with azide via the click reaction (Granzyme B-azide) was obtained. In the control group (Figure 17), the reagents necessary for the click reaction, CuSO4, ascorbic acid, and BTTP, were not added to the reaction solution used for the click reaction. Therefore, the click reaction did not proceed in the control group, and as a result, the active site of Granzyme B was not labeled with biotin. Figure 15 shows the results of evaluating the activity of Granzyme B by detecting luminescence using SDS-PAGE and chemiluminescence after labeling a 0.1 μg / mL Granzyme B-azide solution with ABP using the same method as in the preliminary study of Example 2. Figure 15 shows that when SDS-PAGE and chemiluminescence were used, the band intensity increased depending on the incubation time. This result confirmed that Granzyme B-azide was obtained using the above method.

[0169] [Detection of Granzyme B activity based on the enzymatic activity of the reporter enzyme] Granzyme B-azide obtained after 2 hours of incubation was reacted with anti-Granzyme B antibody-labeled beads prepared in the same manner as in step 2 of Example 2 at concentrations of 0 pg / ml, 100 pg / ml, 200 pg / ml, 300 pg / ml, or 400 pg / ml for 30 minutes at 30°C while shaking at 800 rpm. Thereafter, Granzyme B activity was evaluated based on the enzymatic activity of the reporter enzyme in the same manner as in steps 2 and 3 of Example 2.

[0170] [Results] Figure 16 shows the results of measuring the amount of active Granzyme B in the group (Click+) to which the reagents necessary for the click reaction were added and the group (Click-, control group) to which the reagents necessary for the click reaction were not added in Example 7. Figure 17 shows the percentage (%) of wells in which red fluorescence derived from the metabolite of β-Gal modified with active Granzyme B was observed, among wells in which green fluorescence derived from beads was observed, in the group (Click) to which the reagents necessary for the click reaction were added and the group (Control) to which the reagents necessary for the click reaction were not added in Example 7. According to the results of Figures 16 and 17, a large amount of red fluorescence derived from the metabolite of β-Gal modified with Granzyme B was observed in the group to which the reagents necessary for the click reaction were added, while almost no red fluorescence was observed in the group to which the reagents necessary for the click reaction were not added. These results demonstrate that the method according to an embodiment of the present disclosure can also evaluate the activity of the active site of enzymes other than PSA. Furthermore, according to a method according to one embodiment of the present disclosure, it was shown that the activity of a target protein can be evaluated based on the enzymatic activity of a reporter enzyme not only when the labeling site derived from the activity-based labeling molecule is directly modified with a reporter enzyme, but also when the labeling site is indirectly modified via a linker.

[0171] Example 8: Examination 3 of whether enzyme activity can be detected in plasma It is believed that the activity of granzyme B in blood can change depending on the state of various diseases. Therefore, using liver injury as a model, an examination was conducted to determine whether a method according to an embodiment of the present disclosure can distinguish between healthy mice and liver injury model mice in terms of the proportion of active granzyme B. Ethical approval for research using animals was obtained from the Animal Experimentation Committee of the University of Tokyo (P4-21, P31-9).

[0172] [Creation of Liver Injury Model Mice] Six-week-old male C57BL / 6JJcl mice were purchased from CLEA Japan and acclimatized for 5 days. In the liver injury group, mice were given thioacetamide (TAA, T0817, Tokyo Chemical Industry Co., Ltd.) dissolved in drinking water at a concentration of 300 mg / L to induce liver injury. Meanwhile, healthy control mice were given tap water. After 4 days of feeding, the mice were euthanized, and blood was collected from the inferior vena cava and collected in a 1.5 mL tube containing 1.5 μL of heparin (Yoshitomi Pharmaceutical Co., Ltd.). The collected blood samples were centrifuged at 1700 g and 4°C for 15 minutes, and plasma was separated as the supernatant.

[0173] [Preparation of Measurement Sample and Activity Measurement] The plasma obtained above was diluted 50-fold with HEPES. FP-azide was added to the solution at a concentration of 40 μM and incubated overnight at room temperature. The resulting solution was diluted 10-fold with HEPES. Alkyne-PEG4-biotin (10 μM), CuSO4 (1 mM), ascorbic acid (1 mM), and BTTP (3 mM) were added to the solution and incubated at room temperature for 2 hours. Through the above procedures, azide-labeled Granzyme B (Granzyme B-azide) was obtained via a click reaction. The resulting solution containing Granzyme B-azide was diluted 10-fold with HEPES and reacted with anti-Granzyme B antibody-labeled beads prepared in the same manner as in Step 2 of Example 2 in a 96-well plate at 30°C for 30 minutes while shaking at 800 rpm. Thereafter, the activity of Granzyme B was evaluated based on the enzyme activity of the reporter enzyme in the same manner as in steps 2 and 3 of Example 2.

[0174] [Results] Figure 18 shows the results of evaluating granzyme B activity in liver injury (cholangiocytic injury) model mice and healthy mice in Example 8. Figure 19 shows the percentage (%) of wells in which red fluorescence derived from active granzyme B-modified β-Gal metabolites was observed among wells in which green fluorescence derived from beads was observed in Example 8. Note that the results in Figure 19 are shown as the percentage (%) calculated under each condition minus the percentage (%) calculated from the background results measured without Click Reagent. The results in Figures 18 and 19 indicate that the proportion of active granzyme B was significantly higher in plasma from liver injury model mice than in plasma from healthy mice. These results confirm that a method according to an embodiment of the present disclosure can detect the activity / reactivity of active / reactive sites of proteins in plasma.

Claims

1. A method for evaluating the activity / reactivity of an active / reactive site of a protein, comprising: a labeling step of labeling a protein with an activity-based labeling molecule; and a detection step of detecting, in a solution containing one molecule of the labeled protein, the activity-based labeling molecule that has labeled the protein using an enzymatic reaction of a first reporter enzyme that modifies the activity-based labeling molecule, wherein the activity-based labeling molecule reacts with the active / reactive site of the protein and, after the reaction, forms a covalent bond with the protein or its active / reactive site, and the labeling step includes contacting the activity-based labeling molecule with the protein.

2. The evaluation method according to claim 1, wherein the labeling step further comprises contacting the protein with a post-translational modification labeling molecule, and the detection step comprises detecting the post-translational modification labeling molecule that has labeled the protein in a solution containing one molecule of the labeled protein using an enzymatic reaction of a second reporter enzyme that modifies the post-translational modification labeling molecule, and the post-translational modification labeling molecule is a molecule that selectively binds to a post-translational modification site in the protein.

3. The evaluation method according to claim 1, wherein the labeling step further comprises contacting the protein with an antibody or an antigen-binding fragment thereof that is capable of binding to the protein and that is modified with a third reporter enzyme, and the evaluation method comprises measuring the enzymatic reaction of the third reporter enzyme in a solution containing one molecule of the labeled protein.

4. The evaluation method according to claim 1, wherein the labeling step further comprises contacting the complex with the protein so that one molecule of the protein can bind to each complex of a bead and an antibody or antigen-binding fragment thereof that binds to the protein, and the method further comprises, after the labeling step and before the detection step, placing the complex bound to the protein in microwells so that one bead in the complex can be contained per well.

5. The evaluation method according to claim 4, wherein the beads are fluorescent.

6. The evaluation method according to claim 4, wherein the placing step comprises: pouring an aqueous solution containing a substrate for the first reporter enzyme and a suspension of the complex bound to the protein into a plurality of receptacles of a microchamber array having a hydrophobic surface through openings so that the average number of beads filled in each of the plurality of receptacles is 0.0002 to 0.5; and blocking the openings with a hydrophobic solvent.

7. The evaluation method according to claim 1, wherein the activity-based labeling molecule comprises a reactive site that reacts with the active / reactive site of the protein and a first labeling site, and the labeling step further comprises, after contacting the activity-based labeling molecule with the protein, directly or indirectly modifying the first labeling site derived from the activity-based labeling molecule with the first reporter enzyme.

8. The evaluation method according to claim 1, wherein the active / reactive site is the active center of an enzyme and / or a highly reactive residue of a protein.

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