Application of glyoxal-based compound in preparation of probe

Abstract

The invention relates to the technical field of biology, in particular to application of a glyoxal-based compound in preparation of a probe. According to the invention, the glyoxal-based arginine specific probe is developed, and the reaction activity of arginine in proteome is globally analyzed by utilizing a proteomics analysis method; a fragmented electrophilic small molecule library with a glyoxal structure is constructed, and a new inhibitor is found for protein which cannot be prepared into a medicine through competitive proteomics; and finally, in combination with protein structure calculation and proteome analysis, key ligand arginine for mediating interaction between proteins is found, and the process is disturbed by using inhibitor molecules.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a compound based on glyoxal in preparing a probe. Background Art

[0002] Arginine is an abundant (5.1%) amino acid that plays an important role in protein structure and function. With a strongly basic guanidine group, it is the second most abundant amino acid in protein-protein interaction hotspots. It is worth noting that since the phosphate group on nucleic acids carries a negative charge, arginine is also a key site for mediating protein-DNA or RNA interactions, and plays an important role in cellular processes such as RNA modification and shearing. Arginine is also an important target for post-translational modifications, such as methylation and citrullination. Arginine methylation is mainly catalyzed by the protein arginine methyltransferase (PRMT) family. It mainly occurs on membrane receptors, histones, DNA damage proteins, and RNA binding proteins, and plays an important role in gene transcription, DNA damage repair, RNA shearing, and cancer. In addition, arginine cation-π interactions also play an important role in regulating liquid-liquid phase separation (LLPS), and the methylation state of arginine can regulate the strength of these cation-π interactions. Low-methylated arginine strongly promotes phase separation formation. Citrullination is a key post-translational modification (PTM) that affects protein structure and function. It is catalyzed by human arginine deiminase, converting positively charged arginine into neutral citrulline, leading to changes in intramolecular and extramolecular interactions and protein conformation. Since citrullination severely affects protein structure and folding, some epitopes may change or form new epitopes when proteins are citrullinated, which in turn may trigger immune responses against citrullinated host proteins, leading to the occurrence of autoimmune reactions, rheumatoid arthritis, etc.

[0003] In fact, the reaction of intracellular dicarbonyl compounds with arginine was reported decades ago. After that, 1,2-cyclohexanedione was often used to block arginine on pancreatic enzymes. In addition, methylglyoxal and 2,3-butanedione were used to modify arginine, but these molecules would simultaneously label other nucleophilic amino acids. In contrast, the reaction rate and selectivity of phenylglyoxal on arginine were improved. Wager et al. used azide-modified phenylglyoxal to selectively label arginine on antibodies. Dong developed the arginine-lysine crosslinker KARGO and analyzed the interaction of the complex through crosslinking mass spectrometry results. Therefore, it is feasible to design arginine-specific chemical proteomics reaction probes based on the structure of phenylglyoxal.

[0004] Ye Mingliang's group recently developed a method for dimethylated arginine enrichment based on steric hindrance effects, which enabled a global analysis of arginine dimethylation-regulated phase separation. Phenylacetaldehyde fragments preferentially react with citrulline (over arginine) at acidic pH values ​​to form an imidazolide ring structure. Thompson et al. used biotin-modified phenylglyoxal probes to label citrullination of proteins under acidic conditions (pH = 2-3). Li Lingjun's group reported a method for enriching and identifying citrullination with 2,3-butanedione and biotin thiol tags, and conducted a global study of the citrullinated proteome of mouse tissues.

[0005] In summary, there is still a gap in the research on the reactivity of arginine and its targeting ability by small molecules at the whole proteome level. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, some embodiments of the present invention propose an application of a compound based on glyoxal in preparing a probe.

[0007] In addition, some embodiments of the present invention use a proteomic analysis method based on an arginine-specific probe for glyoxal to perform a global analysis of arginine reactivity in the proteome.

[0008] In addition, some embodiments of the present invention discover new inhibitors for undruggable proteins through competitive screening of fragmented electrophilic small molecule libraries; and use inhibitor molecules to perturb protein-protein interactions.

[0009] In one aspect, the present invention provides the use of a compound based on glyoxal in the preparation of a probe, wherein the compound has a structure shown in Formula I (the two carbonyl carbons in the glyoxal structure are connected to an R1 group and H, respectively):

[0010]

[0011] Wherein, R1 is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms;

[0012] The substitutions are each independently substituted by at least one of halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy, and phenoxy, wherein the number of substitutions ranges from a single substitution to a maximum number of substitutions;

[0013] The probe has the application of any one of A1) to A4):

[0014] A1) Detection or auxiliary detection of arginine;

[0015] A2) preparing a product for detecting or assisting in detecting arginine;

[0016] A3) screening or assisting in screening compounds that bind to arginine;

[0017] A4) Preparation of products for screening or assisting screening of compounds that bind to arginine.

[0018] According to some embodiments of the present invention, the substitutions are each independently substituted by at least one of halogen (e.g., F, Cl, Br), C1-C4 alkyl (e.g., methyl, ethyl), methyl substituted by halogen (e.g., F, Cl, Br), C1-C4 alkoxy (e.g., methoxy, ethoxy), and phenoxy, wherein the number of substitutions is monosubstitution or disubstitution.

[0019] According to some embodiments of the present invention, the substitutions are each independently substituted by at least one of halogen (e.g., F, Cl, Br), C1-C4 alkyl (e.g., methyl, ethyl), F-substituted C1-C4 alkyl (e.g., methyl, ethyl), C1-C4 alkoxy (e.g., methoxy, ethoxy), and phenoxy, wherein the number of substitutions is monosubstitution or disubstitution.

[0020] According to some embodiments of the present invention, the substitutions are each independently substituted by at least one of F, Cl, Br, methyl, trifluoromethyl, methoxy, and phenoxy, wherein the number of substitutions is monosubstitution or disubstitution.

[0021] According to some embodiments of the present invention, the substitutions are each independently monosubstituted by at least one of methyl, trifluoromethyl, methoxy, and phenoxy, or the substitutions are each independently disubstituted by at least one of F, Cl, and Br.

[0022] According to some embodiments of the present invention, R1 is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, naphthyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1 or 2 heteroatoms.

[0023] According to some embodiments of the present invention, R1 is selected from C1-C4 alkyl (eg, methyl, ethyl), substituted or unsubstituted phenyl, naphthyl, furyl.

[0024] According to some embodiments of the present invention, R1 is selected from methyl, substituted or unsubstituted phenyl, naphthyl, furanyl; optionally, the substitutions are each independently substituted by at least one of F, Cl, Br, methyl, trifluoromethyl, methoxy, phenoxy, wherein the number of substitutions is monosubstitution or disubstitution.

[0025] According to some embodiments of the present invention, R1 is selected from methyl, substituted phenyl, naphthyl; optionally, the substitutions are each independently substituted by at least one of F, Cl, Br, methyl, trifluoromethyl, methoxy, and phenoxy, wherein the number of substitutions is monosubstitution or disubstitution.

[0026] According to some embodiments of the present invention, the glyoxal-based compound is selected from:

[0027]

[0028] According to some embodiments of the present invention, the product is selected from a kit, a chip or a system.

[0029] Another aspect of the present invention provides a probe, wherein the probe is obtained by modifying the above-mentioned glyoxal-based compound with a functional group through a click reaction.

[0030] According to some embodiments of the present invention, the functional group of the click reaction is selected from an alkyne group and an azide group, and the functional group is located at the end of the probe.

[0031] According to some embodiments of the present invention, the probe is the above compound connected to a terminal alkyne group or a terminal azide group via 1 to 2 methylene groups.

[0032] According to some embodiments of the present invention, the probe is the R1 group of the above compound connected to the terminal alkyne group or the terminal azide group via 1 or 2 methylene groups.

[0033] According to some embodiments of the present invention, the probe is selected from:

[0034]

[0035] In another aspect, the present invention provides the use of the above probe in any one of A1) to A4),

[0036] A1) Detection or auxiliary detection of arginine;

[0037] A2) preparing a product for detecting or assisting in detecting arginine;

[0038] A3) screening or assisting in screening compounds that bind to arginine;

[0039] A4) Preparation of products for screening or assisting screening of compounds that bind to arginine.

[0040] According to some embodiments of the present invention, the product is selected from a kit, a chip or a system.

[0041] Another aspect of the present invention provides a kit, which comprises the above-mentioned probe.

[0042] According to some embodiments of the present invention, the kit has the use of any one of A1) to A4):

[0043] A1) Detection or auxiliary detection of arginine;

[0044] A2) preparing a product for detecting or assisting in detecting arginine;

[0045] A3) screening or assisting in screening compounds that bind to arginine;

[0046] A4) Preparation of products for screening or assisting screening of compounds that bind to arginine.

[0047] According to some embodiments of the present invention, the product is selected from a kit, a chip or a system.

[0048] In another aspect, the present invention provides a method for detecting or assisting in detecting arginine using the above-mentioned probe, comprising the following steps:

[0049] The probe is mixed with a protein sample and incubated to obtain a first system; the first system is mixed with a first reporter molecule to perform a first click reaction, and a proteomic analysis is performed on the reaction product to detect or assist in detecting arginine in the protein sample;

[0050] Wherein, the first reporter molecule also contains a functional group that can undergo a click reaction with the probe.

[0051] The principle of this method is as follows: the structure of the probe contains a glyoxal reactive group and a functional group for click reaction. Arginine in the protein is a nucleophilic amino acid residue, and there is a guanidine group in its structure, which can react with the glyoxal group in the probe, so that the protein and the probe form a covalently bound complex. The complex is further reacted with a first reporter molecule, wherein the functional group on the probe can undergo a click reaction with the functional group on the first reporter molecule, so that the reporter group is connected to the complex of the protein and the probe for subsequent proteomics analysis.

[0052] The probes used in some embodiments of the present invention are preferably obtained (e.g. ), at the whole proteome level, the probe has excellent identification depth and high selectivity for arginine.

[0053] According to some embodiments of the present invention, the protein sample is a cell lysis supernatant.

[0054] According to some embodiments of the present invention, the mixed incubation is performed at 20-35° C. for 2-6 hours.

[0055] According to some embodiments of the present invention, the first reporter molecule may be derived from a reporter molecule well known in the art for enrichment purposes (e.g., biotin, desthiobiotin) or various reporter molecules for tracing protein purposes (e.g., fluorescent dyes). It should be understood that the "reporter group" is the portion of the "reporter molecule" used for reporting purposes. Examples of fluorescent dyes include, but are not limited to, carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), tetraethylrhodamine, carboxytetramethylrhodamine (TAMRA), fluoroboron fluorescent dyes (BODIPY), cyanine dyes (e.g., Cy3, Cy5, etc.) and Alexa Fluro series dyes (e.g., Alexa Fluro 488, Alexa Fluro 568, etc.). In some preferred embodiments of the present invention, the first reporter molecule may include at least one of biotin and carboxytetramethylrhodamine as a detectable marker. In some embodiments of the present invention, biotin-labeled protein targets can be enriched by binding streptavidin to biotin to facilitate subsequent mass spectrometry analysis.

[0056] According to some embodiments of the present invention, the functional group contained in the first reporter molecule can undergo a click reaction with the functional group modified on the probe, and therefore, the functional group can be selected from an alkyne group and an azido group. It is understood that when the functional group contained in the probe is an alkyne group, the functional group contained in the first reporter molecule is an azido group; and when the functional group contained in the probe is an azido group, the functional group contained in the first reporter molecule is an alkyne group.

[0057] According to some embodiments of the present invention, the reagents used in the first click reaction further include trichloroethyl phosphate, tert-butyl trichloroacetimidate and copper sulfate. According to some embodiments of the present invention, the first click reaction is carried out at 20-35° C. for 0.5-3 h.

[0058] According to some embodiments of the present invention, the first reporter molecule also includes a cleavable group, which includes but is not limited to a photo-cleavable group (PC), an acid-cleavable group (DADPS) and an enzyme-cleavable group (TEV), and the cleavable group is connected to the functional group.

[0059] According to some preferred embodiments of the present invention, in the first reporter molecule, the functional group and the reporter group are connected via a cleavable group. For example, the first reporter molecule has a structural formula selected from the following:

[0060]

[0061] According to some embodiments of the present invention, after the first click reaction is completed, the protein is cleaved by protease to enrich the protein labeled with the first reporter molecule and then cleaved.

[0062] According to some preferred embodiments of the present invention, trypsin is used to perform the enzymatic cleavage.

[0063] According to some preferred embodiments of the present invention, the cleavage is photocleavage.

[0064] According to some preferred embodiments of the present invention, the photolysis is performed under ultraviolet light, and the time of the photolysis is not particularly limited; for example, the photolysis is performed at 365 nm for 0.5 to 2 h to release the peptide segment.

[0065] According to some embodiments of the present invention, the detecting or auxiliary detecting of arginine in the protein sample includes detecting the presence or absence of arginine in the protein sample, and also includes evaluating the reactivity of arginine in the protein sample.

[0066] According to some preferred embodiments of the present invention, the reactivity of arginine is evaluated by comparing the arginine labeling yields at different probe concentrations. For example, the reactivity of arginine in the proteome can be quantified by comparing the primary peptide ion intensity ratio Ratio H / L.

[0067] In another aspect, the present invention provides a method for screening or assisting screening of compounds that bind to arginine using the above-mentioned probe, comprising the following steps:

[0068] contacting the test compound with a protein sample containing arginine to obtain a second system, mixing the second system with the probe and incubating them to obtain a third system; mixing the third system with a second reporter molecule, performing a second click reaction, and analyzing the competitive binding effect of the test compound on arginine in the protein sample;

[0069] Wherein, the second reporter molecule also contains a functional group that can undergo a click reaction with the probe.

[0070] According to some embodiments of the present invention, the contacting is performed at 20-35° C. for 2-6 hours.

[0071] According to some embodiments of the present invention, the quantitative standard of the competitive binding effect is: arginine reacting with the test compound will competitively inhibit the labeling of the probe, resulting in a significant reduction in the degree of labeling. Compared with the DMSO group, the primary peptide ion intensity of the test compound pretreatment group is greatly reduced. The degree of inhibition is reflected by the ligand targeting ratio Ratio DMSO / CP, and the effective ligand is set to R DMSO / CP ≥ 4.

[0072] In addition, the probe, protein sample, mixed incubation conditions, second reporter molecule, reaction conditions of the second click reaction, and steps after the second click reaction are completed can be the same as those in the aforementioned method for detecting or assisting in detecting arginine or can be performed with reference to the aforementioned method, and will not be repeated here.

[0073] Accordingly, using the above-mentioned screening or auxiliary screening methods, some embodiments of the present invention discover new inhibitors for undruggable proteins through the competitive binding effect of fragmented electrophilic small molecule libraries on arginine in protein samples.

[0074] Therefore, another aspect of the present invention provides the use of glyoxal compounds in the preparation of reagents for binding arginine.

[0075] Another aspect of the present invention provides the use of glyoxal compounds in preparing reagents for weakening or inhibiting protein-protein interaction, wherein the protein-protein interaction domain includes arginine.

[0076] According to some embodiments of the present invention, the agent that weakens or inhibits protein-protein interaction is a CDK4-CCND3 interaction inhibitor.

[0077] According to some embodiments of the present invention, the agent that weakens or inhibits protein-protein interaction is a CDK4-CCND1 interaction inhibitor.

[0078] The interaction of CDK4-CCND3 and / or CDK4-CCND1 plays an important role in the occurrence and development of various diseases (such as B cell acute lymphoblastic leukemia, nervous system function, etc., especially in cancer). Therefore, the glyoxal compound has the therapeutic potential for B cell acute lymphoblastic leukemia, nervous system function, etc., especially cancer.

[0079] According to some embodiments of the present invention, the agent that weakens or inhibits protein-protein interaction is a PURA2-PURA1 interaction inhibitor.

[0080] PURA2-PURA1 interaction is associated with the following diseases or conditions: PURA2 and PURA1 genes play an important role in cardiac energy metabolism and function, and the interaction with the cardiac transcription factor GATA4 has an important influence on cardiac gene expression and function. Therefore, the glyoxal compounds have therapeutic potential for diseases related to cardiac gene expression and dysfunction.

[0081] Another aspect of the present invention provides use of glyoxal compounds in the preparation of GSHR inhibitors.

[0082] According to some embodiments of the present invention, the GSHR inhibitor binds to the amino acid at position R81 of GSHR, thereby inhibiting the redox activity of GSHR.

[0083] Another aspect of the present invention provides use of glyoxal compounds in the preparation of KAD1 inhibitors.

[0084] According to some embodiments of the present invention, the KAD1 inhibitor binds to the R97 amino acid of KAD1, thereby inhibiting the interaction between KAD1 and phosphate.

[0085] According to some embodiments of the present invention, the glyoxal compound has a structure shown in formula (II):

[0086]

[0087] wherein R2 or R3 are each independently selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms; or R2 or R3 are connected to form a 6-9 membered spiro ring;

[0088] The substitutions are each independently substituted by at least one of halogen, nitro, hydroxyl, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, and C1-C4 alkoxy, wherein the number of substitutions ranges from a single substitution to a maximum number of substitutions.

[0089] According to some embodiments of the present invention, R2 or R3 are each independently selected from H, substituted or unsubstituted C1-C6 alkyl (methyl, ethyl, propyl, butyl, pentyl), substituted or unsubstituted phenyl, biphenyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1 or 2 heteroatoms; or R2 or R3 are connected to form a 6-9 membered spiro ring.

[0090] According to some embodiments of the present invention, R2 or R3 are each independently selected from H, substituted or unsubstituted C1-C6 alkyl (methyl, ethyl, propyl, butyl, pentyl), substituted or unsubstituted phenyl, biphenyl, furyl, thienyl; or R2 or R3 are connected to form a 6-9 membered spiro ring.

[0091] According to some embodiments of the present invention, the substitutions are each independently substituted by at least one of halogen (e.g., Br, Cl, F), nitro, hydroxyl, C1-C4 alkyl (e.g., methyl, ethyl), halogen-substituted C1-C4 alkyl, and C1-C4 alkoxy (e.g., methoxy), wherein the number of substitutions is monosubstitution or disubstitution.

[0092] According to some embodiments of the present invention, the substitutions are each independently monosubstituted by at least one of halogen, nitro, hydroxyl, methyl, and methoxy.

[0093] According to some embodiments of the present invention, the glyoxal compound is selected from the following structural formula:

[0094]

[0095] According to some embodiments of the present invention, there are at least the following beneficial effects:

[0096] A glyoxal-based arginine-specific probe was developed, and proteomic analysis methods were used to conduct a global analysis of arginine reactivity in the human proteome. New inhibitors were discovered for undruggable proteins through competitive screening of a fragmented electrophilic small molecule library. Finally, inhibitor molecules were used to perturb protein-protein interactions.

[0097] In this specification and claims, reference will be made to a number of terms which shall have the following definitions:

[0098] The term "arginine" includes arginine ( ) or its derivatives, wherein "derivatives thereof" are defined herein as chemical moieties covalently attached to arginine (except the guanidine group on the side chain), such as acids, esters, amides, alcohols, alcoholates, aldehydes, amines, salts, hydrates, glycosides, isomers, enantiomers. It is understood that arginine or its derivatives contain guanidine groups.

[0099] The term "halogen" refers to F, Cl, Br, I.

[0100] The term "alkyl" refers to a straight or branched saturated hydrocarbon group. Non-limiting examples of alkyl include methyl, ethyl, propyl, butyl. Each group includes various isomers, such as propyl includes n-propyl, isopropyl, etc., butyl includes isomers such as n-butyl, isobutyl, sec-butyl, tert-butyl, and pentyl includes n-pentyl, isopentyl, etc. In some embodiments, alkyl can be C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C4 alkyl, C2-C3 alkyl, C3-C4 alkyl.

[0101] The term "halogen-substituted C1-C4 alkyl" refers to a C1-C4 alkyl group substituted by one or more halogen groups, examples of which include fluoromethyl, chloromethyl, 2,2-difluoroethyl, 2,2-dichloroethyl, 3,3-difluoropropyl, 3,3-dichloropropyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 3,3,3-trifluoropropyl, 3,3,3-trichloropropyl, 2,2-difluoropropyl, 3,3-difluorobutyl, 1-bromo-2,2,2-trifluoroethyl, 1-chloro-2,2,2-trifluoroethyl, 1,2,2,2-tetrafluoroethyl, pentafluoroethyl, 2,2,3,3,3-pentafluoropropyl, 1,1,2,2-tetrafluoroethyl and 2,2,3,3-tetrafluoropropyl.

[0102] The term "heteroatom" refers to O, S, N, Se, Si, Ge or the like.

[0103] The term "heteroaryl" refers to a monovalent group of a heterocyclic aromatic system in which at least one carbon atom is replaced by a non-carbon atom based on an aryl group, wherein the non-carbon atom can be selected from O, S, N, Se, Si or Ge. In some embodiments, the heteroaryl group can be pyrrolyl, furanyl, thienyl, pyridyl, pyrazolyl, pyrimidinyl, pyrazinyl, triazinyl, pyridazinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl, thiazolyl, furazanyl, thienyl, etc.

[0104] The term "substituted" in "substituted or unsubstituted" means that one or more hydrogen atoms are replaced by other atoms or functional groups (i.e., substituents), and unless otherwise limited by the definition, it also includes that one or more hydrogen atoms are replaced by a group formed by connecting two or more of the above-mentioned substituents.

[0105] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0107] Figure 1 is a flow chart for identifying arginine sites in the human proteome using probe 1.

[0108] Figure 2 It is a flow chart of probe screening and the result chart, where A: the workflow of probe optimization combining LC-MS / MS with open search, B: the analysis of the number of arginine-modified peptide matching spectra (PSM) in four different groups using PG as the internal standard.

[0109] Figure 3This is a graph showing the results of the investigation of the identification depth and selectivity of probe 1 and probe 2, where A: the modification distribution of probes 1 and 2 was analyzed, considering the case where the peptide matching spectrum number (PSM) value was greater than 80; B: the selectivity of probes 1 and 2 for arginine was evaluated using an open search method; C: the number of modification sites was determined by using a closed search method using the expected modification offset masses of the two probes; D: the selectivity of probes 1 and 2 was determined by an offset search method using the offset masses described in A.

[0110] Figure 4 Figure 1 is a diagram of the optimization of the mass spectrometry sample processing process and the identification of arginine sites in the human proteome, where A: Probe 1 was used to identify arginine labeling sites in membrane proteins and soluble protein fractions, including two biological replicates, B: An alternative process (in which trypsin digestion precedes the enrichment step) was compared with the traditional method (enrichment precedes the digestion step) to identify arginine labeling sites, with two biological replicates, C: A schematic diagram of the optimized workflow, detailing the trypsin digestion before enrichment with probe 1, D: Good reproducibility of labeled arginine sites in two biological replicates, E: Identification of labeled arginine sites using probe 1 in different cell lines, F: Venn diagram of labeled arginine sites in different cell lines, G: Heat map showing the complementarity of labeled arginine sites in different cell lines.

[0111] Figure 5 Figure 1 is a graph of the results of a study of arginine reactivity in the human proteome, where A: a schematic diagram describing the workflow of arginine reactivity analysis, B: a correlation plot showing the log2(RH:L) values ​​in two biological replicates, CH: in the HEK293T cell proteome, the recombinant wild-type protein and its corresponding arginine to alanine mutant were treated with probe 1, and protein labeling containing specific arginine reactivity was verified by gel fluorescence.

[0112] Figure 6 It is a graph of the results of the chemical property study of the molecular library, wherein: A: a comparison of the cLogP value of the compound and the molecular weight, showing the glyoxal compounds that conform to the drug-like five rules (Ro5, light gray box) and the lead compound-like three rules (Ro3, dark gray box); B: distribution of glyoxal compounds based on the number of hydrogen bond donors (HBDs), hydrogen bond acceptors (HBAs) and rotatable bonds (RBs), highlighting the compounds that follow Ro5 (light gray box) and Ro3 (dark gray box); C: reactivity analysis of glyoxal compounds (0.5-10 mM) in a pH 7.8 buffer (50 mM borate / 30% acetonitrile) by incubation at 37°C for 4 hours with nucleophiles Nα-acetyl-L-arginine, cysteine, lysine or histidine methyl ester (50-100 mM).

[0113] Figure 7 Figure 1 is a graph of the results of ligand-targeted studies in the human proteome, wherein: A: a schematic diagram describing the competitive DIA-ABPP workflow for identifying arginine bound to glyoxal compounds; B: the ratio of liganded protein (left) and liganded arginine (right) compared to quantified protein and arginine; C: WB analysis of probe 1 labeling after pretreatment with compound 1 in HEK293T cell lysates expressing wild-type and arginine to alanine mutant GSHR; D: a bar graph showing the effect of compound 1 and R81A mutation on GSHR redox-dependent enzyme activity; E: WB analysis of probe 1 labeling after pretreatment with compound 1 in HEK293T cell lysates expressing wild-type and arginine to alanine mutant KAD1; F: a bar graph showing the effect of compound 1 on KAD1 kinase activity.

[0114] Figure 8 This is a study result of targeting key arginine to interfere with protein-protein interactions, where A: The schematic diagram shows the change in the solvent accessible surface area (SASA) of ligand arginine when the complex is formed, B: The heat map shows the ligand arginine that may mediate protein-protein interactions. The SASA values ​​of these arginines are reduced by at least 50% after the formation of binary protein complexes, CH: Representative co-immunoprecipitation experiments verify the disruption of selected protein-protein interactions by fragmented small molecules; The bar graph shows the protein abundance quantification of WB analysis, and the data are presented as the mean ± standard deviation of three biological replicates. DETAILED DESCRIPTION

[0115] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0116] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0117] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0118] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0119] Unless otherwise specified, "about" in the present invention means that the allowable error is within ±5%.

[0120] Some embodiments of the present invention develop arginine-specific probes 1 based on phenylglyoxal and use proteomics analysis methods to perform a global analysis of the reactivity of arginine in the human proteome. In addition, some embodiments of the present invention discover new inhibitors for undruggable proteins through competitive screening of fragmented electrophilic small molecule compound libraries; finally, the inhibitor molecules are used to perturb protein-protein interactions.

[0121] The specific methods include: screening from a focused molecular library of 13 phenylglyoxal derivatives and adding an azide tag to obtain an arginine-specific probe 1; through the optimization of reaction conditions and the process of proteomic research, the arginine-specific probe 1 containing the azide tag is mixed with the protein lysate, and then a click reaction is carried out with an acetylenic reagent containing a biotin tag, and finally a proteomic analysis is performed to obtain arginine sites with different reactivity; a commercially available fragmented electrophilic small molecule library with a glyoxal structure is purchased, and new inhibitors are discovered for undruggable proteins through competitive proteomics; finally, protein structure calculation and proteomic analysis are combined to find the key ligand arginine that mediates protein-protein interactions, and inhibitor molecules are used to perturb this process.

[0122] Example 1

[0123] In this example, a phenylglyoxal-based arginine-specific probe 1 (compound B3 plus an azide tag) was developed, and the screening process included the following steps:

[0124] The glyoxal derivatives purchased from Bidex Pharmaceuticals were selected to form a focused molecular library of 13 molecules:

[0125]

[0126] Dissolve 13 small molecules (PG, A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3) in DMSO and prepare 200mM stock solution. There are 13 small molecules in total, 4 in a group, PG molecule is used as the internal standard in each group, 10μL (PG, A1, A2, A3) is mixed into Group_A (the concentration of each molecule is 50mM), and so on, 10μL (PG, B1, B2, B3) is mixed into Group_B (50mM), 10μL (PG, C1, C2, C3) is mixed into Group_C (50mM), and 10μL (PG, D1, D2, D3) is mixed into Group_D (50mM).

[0127] Six proteins (RNaseA, catalase, Lysozyme, lactoferrin, aldolase and β-amylase) were dissolved in PBS and prepared into 2 mg / mL stock solutions. In 100 μL PBS system, 7.14 μL of protein stock solution was added to prepare 1 mg / mL protein mixed solution.

[0128] Then take 2μL of 50mM A, B, C, D small molecule mixture and add it to 100μL protein mixture to react for 2 hours. Mix the resulting system with 5 times the volume of pre-cooled acetone for precipitation, and dissolve it with 60μL of 25mM ABC (ammonium carbamate) containing 6M Urea (urea). Next, the resulting system is reduced with DTT (dithiothreitol) at a final concentration of 10mM, 55°C for 40 minutes and cooled to room temperature. Add IAA (iodoacetamide) at a final concentration of 15mM, place it on a rotary mixer, and alkylate at room temperature in the dark for 30 minutes. Add DTT solution at a final concentration of 5mM to quench the IAA reaction (place it on a rotary mixer at room temperature for 10 minutes). Next, 300 μL of 25 mM ABC was added to dilute the concentration of Urea in the obtained system to 1 M, and CaCl2 with a final concentration of 1 mM (calcium ions are used to increase trypsin trypsin activity), 1:100 trypsin (i.e., 1 μg trypsin is added to 100 μg of protein group), and shaken at 37 ° C overnight (1050 rpm, about 12 hours). Finally, the obtained system was placed on ice, and 20% (volume fraction) formic acid was slowly added to adjust the pH to 2-3 (monitored by pH test paper) to quench the trypsin digestion reaction. Finally, a 96-well desalting plate (which can tolerate 20 μg peptides, 200 μL system) was used for desalting, and 0.1% (v / v) formic acid was added for re-dissolution, injection, and LC-MS detection was performed. The mass spectrometry data was searched using a database.

[0129] Open search results are as follows Figure 1As shown, B3 and D1 have the largest number of peptide matching spectra (PSM) containing arginine modification, reaching 3.0 times and 2.2 times relative to the PG internal standard. Therefore, B3 and D1 were selected to add azide tags to synthesize probes 1 and 2.

[0130] The synthesis steps of probe 1 are as follows:

[0131]

[0132] Synthesis Step 1: Synthesis of 1-(4-(p-tolyloxy)phenyl)ethane-1-one:

[0133]

[0134] 1-(4-Fluorophenyl)ethane-1-one (10.0 g, 72.390 mmol, 1.0 eq) and p-methylphenol (7.83 g, 72.390 mmol, 1.0 eq) were dissolved in N,N-dimethylformamide (70 mL, 7V), followed by the addition of potassium carbonate (20.02 g, 144.780 mmol, 2.0 eq), and the mixture was heated to 100°C with stirring and reacted at this temperature for 16 hours. The mixture was cooled to room temperature, ethyl acetate (100 mL) and water (300 mL) were added, and the mixture was stirred to separate layers. The separated aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, and then washed with saturated brine (50 mL), and concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) to obtain the target product (6.60 g, 40.5%) as a light brown solid.

[0135] Synthesis of probe 1 Step 2: Synthesis of 1-(4-(4-(bromomethyl)phenoxy)phenyl)ethan-1-one:

[0136]

[0137] 1-(4-(p-Tolyloxy)phenyl)ethane-1-one (6.50 g, 28.725 mmol, 1.0 eq) and N-bromosuccinimide (5.62 g, 31.598 mmol, 1.1 eq) were dissolved in carbon tetrachloride (65 mL, 10 V), followed by the addition of dibenzoyl peroxide (650.0 mg, 10% wt), the temperature was raised to reflux, and the mixture was stirred for 16 hours under reflux. The mixture was cooled to room temperature, water (50 mL) was added, the mixture was stirred and separated, the separated aqueous phase was extracted with dichloromethane (50 mL), and the organic phases were combined. The organic phase was washed with saturated brine (40 mL), the organic phase obtained by separation was concentrated to dryness, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain the target product (5.52 g, 62.9%) as an off-white solid.

[0138] Synthesis of Probe 1 Step 3: Synthesis of 1-(4-(4-(azidomethyl)phenoxy)phenyl)ethan-1-one:

[0139]

[0140] 1-(4-(4-(bromomethyl)phenoxy)phenyl)ethan-1-one (5.50 g, 16.384 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (55 mL, 10 V), followed by potassium carbonate (4.53 g, 32.768 mmol, 2.0 eq), and then sodium azide (1.28 g, 19.661 mmol, 1.2 eq) was added in batches. After the addition was completed, the mixture was stirred at room temperature for 3 hours. Water (200 mL) and ethyl acetate (55 mL) were added, and the layers were separated by stirring. The separated aqueous phase was extracted with ethyl acetate (35 mL), and the organic phases were combined, washed with saturated brine (35 mL), and concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to obtain the target product (4.10 g, 85.1%) as an oil.

[0141] Synthesis of probe 1 Step 4: Synthesis of (1-(4-(4-(azidomethyl)phenoxy)phenyl)vinyl)oxy)trimethylsilane:

[0142]

[0143] 1-(4-(4-(azidomethyl)phenoxy)phenyl)ethane-1-one (4.10 g, 15.339 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (60 mL, 15 V), cooled to 0 ° C under nitrogen protection, and the temperature was controlled below 5 ° C. Trimethylsilyl trifluoromethanesulfonate (4.10 g, 18.408 mmol, 1.2 eq) was added dropwise, followed by triethylamine (2.33 g, 23.008 mmol, 1.5 eq). After the addition was complete, the mixture was transferred to room temperature for reaction for 3 hours. Water (60 mL) was added, and the layers were stirred and separated. The separated aqueous phase was extracted with dichloromethane (30 mL), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the target product (5.40 g, N / A) as a light brown oil, which was directly used in the next step.

[0144] Synthesis of Probe 1 Step 5: Synthesis of 1-(4-(4-(azidomethyl)phenoxy)phenyl)-2-((trimethylsilyl)oxy)ethan-1-one:

[0145]

[0146] Dissolve (1-(4-(4-(azidomethyl)phenoxy)phenyl)vinyl)oxy)trimethylsilane (5.40 g, 15.339 mmol, 1.0 eq, crude product from the previous step, calculated as 100% yield) in anhydrous dichloromethane (29 mL, 5 V), add a solution of m-chloroperbenzoic acid (3.97 g, 23.009 mmol, 1.5 eq) in dichloromethane (58 mL) dropwise under nitrogen protection, and after the addition is complete, transfer to room temperature and stir for 5 hours. Add saturated sodium bicarbonate and saturated sodium thiosulfate solutions to quench the reaction. Stir and separate, extract the separated aqueous phase with dichloromethane (30 mL), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, and concentrate to dryness to obtain the target product (5.70 g, N / A) as an oil, which is directly used in the next step.

[0147] Synthesis of Probe 1 Step 6: Synthesis of 1-(4-(4-(azidomethyl)phenoxy)phenyl)-2-hydroxyethane-1-one:

[0148]

[0149] 1-(4-(4-(azidomethyl)phenoxy)phenyl)-2-((trimethylsilyl)oxy)ethane-1-one (5.70 g, 15.339 mmol, 1.0 eq, crude product from the previous step, calculated as 100% yield) was dissolved in anhydrous tetrahydrofuran (57 mL, 10 V), and 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (18.41 mL, 18.407 mmol, 1.2 eq) was added dropwise. After the addition was completed, the mixture was stirred at room temperature for 3 hours. Most of the tetrahydrofuran was concentrated under reduced pressure, and ethyl acetate (50 mL) and water (50 mL) were added. The mixture was stirred and separated into layers, and the separated aqueous phase was extracted with ethyl acetate (30 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was chromatographed on a silica gel column (PE:EA=5:1) to obtain the target product as a light yellow solid (1.06 g, 24.5%).

[0150] Synthesis of Probe 1 Step 7: Synthesis of 2-(4-(4-(azidomethyl)phenoxy)phenyl)-2-oxoacetaldehyde:

[0151]

[0152] 1-(4-(4-(azidomethyl)phenoxy)phenyl)-2-hydroxyethane-1-one (5.5 g, 19.415 mmol, 1.0 eq) was dissolved in ethyl acetate (110 mL, 20 V), followed by the addition of 2-iodobenzoic acid (10.87 g, 38.829 mmol, 2.0 eq), heated to reflux and stirred for two hours. After cooling, the mixture was filtered on diatomaceous earth, and the filtrate was concentrated to dryness. The crude product was chromatographed on a silica gel column (PE:EA=2:1) ​​to obtain the target product as an off-white solid (800.0 mg, yield 14.7%). 1 H NMR (500MHz, DMSO) δ8.11(d,J=8.8Hz,2H),7.45(d,J=8.3Hz,2H),7.15(d,J=8.4Hz,2 H), 7.07 (d, J = 8.8Hz, 2H), 6.69 (d, J = 7.1Hz, 2H), 5.64 (t, J = 7.1Hz, 1H), 4.48 (s, 2H). MS(ESI,neg.ion)m / z:296[MH] - .

[0153] The synthesis steps of probe 2 are as follows:

[0154]

[0155] Synthesis Step 1: Synthesis of 1-(6-methylnaphthalen-2-yl)ethan-1-one

[0156]

[0157] Add nitrobenzene (56mL, 8V) to the reaction bottle, cool to about 5°C, add aluminum chloride (7.97g, 59.916mmol, 1.2eq), and stir until dissolved. Control the temperature below 5°C, add acetyl chloride (4.34g, 54.932mmol, 1.1eq), add the mixture and stir for 0.5 hours. In another reaction bottle, dissolve 2-methylnaphthalene (7.10g, 49.930mmol, 1.0eq) in nitrobenzene (28mL, 4V), and cool to 0°C while stirring. Control the temperature below 5°C, add the prepared acetylation reagent dropwise to the nitrobenzene solution of 2-methylnaphthalene, add the mixture dropwise and react for 2 hours at a temperature below 5°C. The reaction solution was added dropwise to a solution of ethanol (10 mL) and water (70 mL), stirred and separated into layers, the separated aqueous phase was extracted with ethyl acetate (70 mL), the organic phases were combined and washed with saturated brine, and concentrated to dryness. The crude product was chromatographed on a silica gel column (PE: EA = 100: 1) to give the target product as an off-white solid (5.20 g, 56.5%).

[0158] Synthesis of Probe 2 Step 2: Synthesis of 1-(6-(bromomethyl)naphthalen-2-yl)ethan-1-one

[0159]

[0160] 1-(6-methylnaphthalene-2-yl)ethane-1-one (5.20 g, 28.224 mmol, 1.0 eq) and N-bromosuccinimide (5.53 g, 31.046 mmol, 1.1 eq) were dissolved in carbon tetrachloride (52 mL, 10 V), followed by the addition of dibenzoyl peroxide (520.0 mg, 10% wt), the temperature was raised to reflux, and the mixture was stirred for 16 hours under reflux. The mixture was cooled to room temperature, water (52 mL) was added, and the mixture was stirred to separate into layers. The separated aqueous phase was extracted with dichloromethane (35 mL), the organic phases were combined, and the organic phases were washed with saturated brine (35 mL). The organic phase obtained by separation was concentrated to dryness, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain the target product (4.86 g, 65.5%) as an off-white solid.

[0161] Synthesis of Probe 2 Step 3: Synthesis of 1-(6-(azidomethyl)naphthalen-2-yl)ethan-1-one

[0162]

[0163] 1-(6-(Bromomethyl)naphthalen-2-yl)ethane-1-one (4.80 g, 18.242 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (48 mL, 10 V), followed by potassium carbonate (4.53 g, 36.484 mmol, 2.0 eq), and then sodium azide (1.42 g, 21.890 mmol, 1.2 eq) was added in batches. After the addition was completed, the mixture was stirred at room temperature for 3 hours. Water (196 mL) and ethyl acetate (50 mL) were added, and the layers were stirred. The separated aqueous phase was extracted with ethyl acetate (30 mL), and the organic phases were combined, washed with saturated brine (30 mL), and concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to obtain the target product (3.42 g, 83.2%) as an oil.

[0164] Synthesis of Probe 2 Step 4: Synthesis of (1-(6-(azidomethyl)naphthalen-2-yl)vinyl)oxy)trimethylsilane

[0165] 1-(6-(Azidomethyl)naphthalen-2-yl)ethane-1-one (3.40 g, 15.094 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (51 mL, 15 V), cooled to 0 ° C under nitrogen protection, and the temperature was controlled below 5 ° C. Trimethylsilyl trifluoromethanesulfonate (4.03 g, 18.113 mmol, 1.2 eq) was added dropwise, followed by triethylamine (2.29 g, 22.641 mmol, 1.5 eq). After the addition was complete, the mixture was transferred to room temperature for reaction for 3 hours. Water (51 mL) was added, and the layers were stirred and separated. The separated aqueous phase was extracted with dichloromethane (20 mL), and the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the target product (4.70 g, N / A) as a light brown oil. The crude product was directly used for the next step.

[0166] Synthesis of Probe 2 Step 5: Synthesis of 1-(6-(azidomethyl)naphthalen-2-yl)-2-((trimethylsilyl)oxy)ethan-1-one

[0167]

[0168] Dissolve (1-(6-(azidomethyl)naphthalene-2-yl)vinyl)oxy)trimethylsilane (4.70 g, 15.094 mmol, 1.0 eq, crude product from the previous step, calculated as 100% yield) in anhydrous dichloromethane (24 mL, 5 V), add a solution of m-chloroperbenzoic acid (3.91 g, 22.641 mmol, 1.5 eq) in dichloromethane (47 mL, 10 V) dropwise under nitrogen protection, and after the addition is complete, transfer to room temperature and stir for 5 hours. Add saturated sodium bicarbonate and saturated sodium thiosulfate solutions to quench the reaction. Stir and separate, extract the separated aqueous phase with dichloromethane (30 mL), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, and concentrate to dryness to obtain the target oily product (4.98 g, N / A), which is directly used in the next step.

[0169] Synthesis of Probe 2 Step 6: Synthesis of 1-(6-(azidomethyl)naphthalen-2-yl)-2-hydroxyethan-1-one

[0170]

[0171] Dissolve 1-(6-(azidomethyl)naphthalen-2-yl)-2-((trimethylsilyl)oxy)ethane-1-one (4.98 g, 15.094 mmol, 1.0 eq, crude product from the previous step, calculated as 100% yield) in anhydrous tetrahydrofuran (50 mL, 10 V), add 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (18.2 mL, 18.113 mmol, 1.2 eq) dropwise, and stir at room temperature for 3 hours after the addition is complete. Concentrate most of the tetrahydrofuran under reduced pressure, add ethyl acetate (50 mL) and water (50 mL). Stir and separate, extract the separated aqueous phase with ethyl acetate (30 mL), combine the organic phases, wash with saturated brine (30 mL), dry with anhydrous sodium sulfate, filter, and concentrate to dryness. The crude product obtained is chromatographed on a silica gel column (PE:EA=5:1) to obtain the target product as a light yellow solid (746.5 mg, 20.5%).

[0172] Synthesis of Probe 2 Step 7: Synthesis of 2-(6-(azidomethyl)naphthalen-2-yl)-2-oxoacetaldehyde

[0173]

[0174] 1-(6-(Azidomethyl)naphthalen-2-yl)-2-hydroxyethane-1-one (5.0 g, 20.725 mmol, 1.0 eq) was dissolved in ethyl acetate (100 mL, 20 V), followed by the addition of 2-iodobenzoic acid (11.61 g, 41.451 mmol, 2.0 eq), heated to reflux and stirred for two hours. The mixture was cooled to room temperature, filtered on celite, and the filtrate was concentrated to dryness. The crude product was chromatographed on a silica gel column (PE: EA = 2: 1) to obtain the desired product as a light brown solid (562.0 mg, yield 11.3%). 1 H NMR (500MHz, DMSO) δ8.78(s,1H),8.14(d,J=8.5Hz,1H),8.10(d,J=8.6,1.3Hz,1H),8.05(d,J=8 .4Hz,1H),7.99(s,1H),7.60(d,J=8.4Hz,1H),6.79(d,J=6.8Hz,2H),5.82(s,1H),4.69(s,2H). MS(ESI,neg.ion)m / z:254[MH] - .

[0175] Next, this example uses mass spectrometry to compare the identification depth and selectivity of probe 1 and probe 2 at the whole proteome level. Figure 2As shown, the results showed that probe 1 had higher selectivity for labeling arginine than probe 2, and probe 1 could identify about 1,600 sites, while probe 2 could only identify about 600 sites. Therefore, probe 1 was selected as the arginine-specific probe for subsequent proteomics research.

[0176] Example 2

[0177] This example develops a method for identifying arginine sites in the human proteome using probe 1, and the process is as follows: Figure 3 As shown, the specific steps include:

[0178] HeLa cells were seeded in 15 cm culture dishes and washed twice with pre-cooled PBS when the cell density reached 90%, scraped and resuspended in 50 mM boric acid / HEPES buffer containing EDTA-free protease inhibitors. Ultrasonic disruption was performed for 1 minute, and the debris was removed by centrifugation at 14,000 g for 10 minutes at 4 °C. The lysate was quantified by BCA to 1.5 mg / mL. 4 mL of lysate was incubated with 1 mM probe 1 at room temperature for 4 hours. Then, the cells were incubated with photocleavable alkyne-biotin CAS No.: 1869922-24-6) for click reaction (room temperature for 1 hour, 0.1 mM alkyne-biotin, 1 mM trichloroethyl phosphate (TCEP) reducing agent, 0.1 mM tert-butyl trichloroacetimidate (TBTA) ligand and 1 mM CuSO4). After the click reaction, the reaction solution was mixed with a precipitation solution (methanol and chloroform mixture) according to methanol / chloroform / reaction solution = 4 mL: 1 mL: 3 mL, and centrifuged at 4,500 g for 10 minutes at room temperature, the supernatant was discarded, the precipitate was washed with 1 mL of methanol, and reconstituted with 25 mM ammonium carbamate (ABC) buffer containing 8 M urea, 25 mM ABC containing 10 mM dithiothreitol (DTT) was added, reduced at 55°C for 40 minutes, and alkylated with 15 mM fresh iodoacetamide (IAA) at room temperature in the dark for 30 minutes. Next, the obtained system was mixed with the precipitation solution according to the ratio of pre-cooled acetone / obtained system = 5mL:1mL, and centrifuged at 4,500g for 10 minutes at room temperature. The supernatant was discarded, the precipitate was washed with 1mL acetone, and re-dissolved with 2mL 25mM ammonium carbamate (ABC) containing 1M urea, and 1mM CaCl2 and 100μg trypsin were added to digest overnight at 37°C with shaking. Then, the obtained system was diluted with 2mL PBS and incubated with 400μL streptavidin magnetic beads at room temperature for 4 hours. The beads were washed three times with 5mL PBS containing 0.2% SDS, three times with 5mL PBS containing 1M urea, and three times with 5mL PBS. Finally, after washing three times with 1mL distilled water, the magnetic beads were resuspended in 600μL 70% methanol water in a 24-well plate, placed on ice, and irradiated with 365nm light for 60 minutes to release the peptides. The supernatant in the 24-well plate was collected in a 1.5 mL low-binding EP tube, and the magnetic beads in the 24-well plate were washed twice with 400 μL of 70% methanol water, and the combined samples were dried in a SpeedVac vacuum concentrator. For LC-MS / MS analysis, the samples were re-dissolved with 0.1% (v / v) formic acid water and analyzed using a QE Plus mass spectrometer with a nano-spray ionization (nano-ESI) source and Xcalibur4.3 software.

[0179] Example 3

[0180] This embodiment provides a method for optimizing the arginine site identification process using probe 1, and the process is as follows: Figure 4 As shown, the specific steps include:

[0181] First, this embodiment compares the membrane plasma component classification treatment with the non-classification treatment in the site identification process. The membrane plasma component classification process is as follows: scrape HeLa cells and resuspend them in 50mM boric acid / HEPES buffer, ultrasonically disrupt for 1 minute, use an ultracentrifuge to separate the cell membrane component and the plasma component, 100,000g, 4°C centrifuge for 45 minutes, and collect the supernatant into a new EP tube. Add 400μL of 50mM boric acid / HEPES solution containing 0.5% Triton X-100 to the precipitate, and ultrasonicate for 1 to 2s to dissolve it again. After BCA quantification, adjust it to the same concentration and refer to the steps in Example 2: use probe 1 to treat the membrane and plasma components, click reaction to connect alkynyl-biotin, trypsin digestion, streptavidin magnetic bead enrichment, photo-cutting, and LC-MS / MS identification.

[0182] Next, (on the basis of non-grading treatment), different enrichment methods in the site identification process are compared, and the process of enrichment followed by enzymatic treatment is as follows: refer to the steps in Example 2: using probe 1 to treat the HeLa lysate, click reaction to connect alkynyl-biotin, streptavidin magnetic beads enrichment, trypsin digestion, photo-cutting, LC-MS / MS identification; the process of enzymatic hydrolysis followed by enrichment is as follows: refer to the steps in Example 2: using probe 1 to treat the HeLa lysate, click reaction to connect alkynyl-biotin, trypsin digestion, streptavidin magnetic beads enrichment, photo-cutting, LC-MS / MS identification.

[0183] The results are as follows Figure 4 As shown in the figure, the study found that the number of sites identified by fractionation of membrane plasma components was comparable to that of unfractionation. In addition, by comparing different enrichment methods during sample processing, it was found that more arginine sites were identified by enzymatic digestion followed by enrichment: HeLa lysate was treated with probe 1, click reaction was used to connect alkynyl-biotin, trypsin digestion, streptavidin magnetic beads enrichment, and photo-cleavage. LC-MS / MS identified about 2,500 sites, about 3 times more than the initial PG probe, and multiple biological replicates were performed to identify the repeatability of the labeling.

[0184] Finally, referring to the above steps of Example 3, only HeLa cells were replaced with Ramos cells, MDA-MB-231 cells or A549 cells, so as to mark and identify a variety of different cell lines. Consistent with existing literature reports, the proteome composition of the MDA-MB-231 cell line and the Ramos cell line is complementary, so a total of 4606 arginine sites were identified in different cell lines, which also verified the complementarity of 231 cells and Ramos cells.

[0185] Example 4

[0186] This embodiment provides a method for globally analyzing the reactivity of arginine in the human proteome using probe 1, and its process is as Figure 5 shown, specifically including the following steps:

[0187] In this embodiment, the reactivity of arginine is evaluated by comparing the arginine labeling yields at different probe concentrations. Arginines with high reactivity will obtain almost equal labeling yields; however, arginines with low reactivity will exhibit probe concentration-dependent labeling yields. The quantitative proteome method of reductive dimethylation (ReDiMe) is used to perform different isotope labelings on the samples treated with two probe concentrations respectively.

[0188] Referring to the steps in Example 2, first, use high and low concentrations of the probe (1 mM and 100 μM) to label the lysate at room temperature for 4 hours, perform click reaction at room temperature for 1 hour, digest with trypsin overnight at 37 °C, enrich with streptavidin magnetic beads at room temperature for 4 hours, then wash the magnetic beads twice with 5 mL of PBS, twice with 5 mL of deionized water, twice with 5 mL of 100 mM TEAB buffer, and then resuspend in 100 μL of 100 mM TEAB buffer. Perform heavy dimethylation labeling on the magnetic beads labeled with high-concentration probe: add 8 μL of 4% D 13 CDO and 8 μL of 0.6 M NaBD3CN on ice; perform light dimethylation labeling on the magnetic beads labeled with low-concentration probe: add HCHO and NaBH3CN. After reacting at room temperature for 2 hours, wash the magnetic beads twice with 100 mM TEAB buffer, combine, and wash three times with 5 mL of H2O, perform photocleavage on ice for 1 hour, and finally analyze the reactivity of arginine by LC-MS / MS. Whether it is a high-concentration or low-concentration probe, it will preferentially react with arginines with high reactivity, and the remaining high-concentration probe will react with arginines with low reactivity. Therefore, by comparing the ratio of the first-order peptide ion intensities "reactivity ratio, RH / L", the reactivity of arginine in the whole proteome can be quantified, corresponding to 0.5 < RH:L < 2 (high reactivity), 2 < RH:L < 4 (medium reactivity), and RH:L > 4 (low reactivity) respectively.

[0189] Next, the reactivity of the arginine site was verified by in-gel fluorescence experiment. HEK293T cells were seeded in 6 cm culture dishes. After 24 hours, 5 μg of wild-type and mutant plasmids were transfected into the cells using 8 μL of Lipofectamine 8000 transfection reagent. About 24 hours after transfection, the cells were lysed by ultrasound with 50 mM boric acid / HEPES buffer, and BCA was quantified to 1.5 mg / mL. Probe 1 was added to 300 μL of lysate at a final concentration of 200 μM and incubated at room temperature for 4 hours. Then 30 μL of pre-washed Flag magnetic beads were added, enriched at 4°C overnight, and then 30 μL of the prepared 1.5 mg / mL Flag peptide solution was added, and competition was carried out at 4°C for 1 hour and at room temperature for 1 hour. The Flag magnetic beads were adsorbed on a magnetic stand and the supernatant was collected. Finally, prepare 30 μL of click reaction cocktail: TCEP (50 mM, final concentration 1 mM); TBTA ligand (1.7 mM, final concentration 100 μM); CuSO4 (50 mM, final concentration 1 mM), TAMRA-Alkyne (carboxytetramethylrhodamine (TAMRA) alkyne, 10 mM, final concentration 100 μM), mix well, incubate at room temperature for 1 hour. Add 10 μL 4× Loading buffer and boil for 10 minutes for SDS-PAGE analysis.

[0190] This example uses an arginine-specific probe 1 for proteomics analysis, and performs a global analysis of the arginine reactivity in the human proteome: using high and low concentrations of probe 1 to label samples, click reaction, trypsin digestion, streptavidin magnetic bead enrichment, and then introducing isotope-labeled dimethyl modification to high and low concentration samples, mixing, photo-cutting, and finally identifying the reactivity of arginine by LC-MS / MS. Both high-concentration and low-concentration probes will preferentially react with highly active arginine, so the reactivity of arginine in the entire proteome can be quantified by comparing the primary peptide ion intensity ratio Ratio H / L. Among all the identified arginine sites, the vast majority showed probe concentration-dependent labeling, which means that these sites are low-active, and only a small number of identified arginine sites showed high reactivity.

[0191] like Figure 5As shown, in this example, several proteins with highly reactive arginine were selected, and the proteins with highly reactive arginine were verified using the targeted mass spectrometry method of parallel reaction monitoring (PRM), including R81 of GSHR (GeneID: 2936), R284 of RIR1 (GeneID: 6240), and R217 of FSCN1 (GeneID: 6624); the primary chromatographic peaks were extracted from the original data by Skyline for targeted quantitative determination, and subsequent in-gel fluorescence experiments also proved that the highly reactive arginine mutation resulted in a significant reduction in probe labeling.

[0192] In addition, this example uses a targeted mass spectrometry method of parallel reaction monitoring (PRM) to verify proteins with low reactivity to arginine, including R115 of FKBP2 (GeneID: 2286), R29 of KT3K (GeneID: 79672), R48 of PARK7 (GeneID: 11315), and R37 of CAPZA1 (GeneID: 829); mutation of low-reactivity arginine in proteins with several reactive arginines results in negligible changes in labeling, while mutation of proteins with only a single reactive arginine, regardless of low or moderate reactivity, results in blocking of labeling.

[0193] Example 5

[0194] This embodiment provides a method for globally analyzing arginine targeting in the human proteome through competitive screening of a fragmented electrophilic small molecule library to discover new inhibitors for undruggable proteins.

[0195] This example designs 32 electrophilic fragment molecules targeting arginine based on the common pharmacophore structure in drug molecules, and then uses the "competitive proteomics" method to evaluate the small molecule targeting of arginine. Specifically, a library of glyoxal molecules with different substituents was first purchased, and the glyoxal molecule library is divided into three chemical types, namely aromatic substitution, alkyl substitution and monosubstitution.

[0196]

[0197] Next, the chemical properties of the small molecule library were explored. It was found that most of the molecules met the five principles of drug-like compounds and the three principles of lead compounds (such as Figure 6 Finally, the reaction specificity of the molecular library was studied. According to literature reports, glyoxal compounds mainly react with the side chains of arginine, cysteine, lysine and histidine. Therefore, the amino and carboxyl ends of the four amino acids were protected to obtain model amino acids, which reacted with the molecules in the molecular library respectively.

[0198] The method for global analysis of arginine targeting in human proteome in this embodiment is carried out with reference to the steps in Example 2, such as Figure 7 As shown in , the proteome was pretreated with fragmentation molecules or DMSO at room temperature for 4 hours, then incubated with probe 1 at room temperature for 4 hours, clicked at room temperature for 1 hour, digested with trypsin at 37°C overnight, enriched with streptavidin magnetic beads at room temperature for 4 hours, and optically cut off on ice for 1 hour. Finally, the ligand targeting of arginine was analyzed by LC-MS / MS based on DIA mode. Figure 7 As shown in , 526 proteins that can be targeted by ligands and 687 arginines that can be targeted by ligands were identified. Arginines that react with fragmented molecules will competitively inhibit the labeling of the probe, resulting in a significant reduction in the degree of labeling (>75%). Compared with the DMSO control group, the ratio of the primary peptide ion intensity is greatly reduced. The degree of inhibition is reflected by the "ligand targeting ratio, RDMSO / CP", and the effective ligand is set to RDMSO / CP ≥ 4. Figure 6 As shown in , LC-MS results show that all molecules do not react with lysine K and histidine H, and a small part reacts with cysteine ​​C to form unstable hemiacetal.

[0199] Next, in this example, each identified arginine that can be targeted by a ligand and each ligand were plotted into a heat map for structure-activity relationship analysis, and it was found that compound 1 (CP1) targets R81 of GSHR. Then, R81 of GSHR was mutated, and the results were as expected, as shown in Figure 7 As shown in Figure C, compound 1 can block the labeling of the wild-type WT, while the labeling of the mutant R81A is weakened, and compound 1 has no effect on the labeling of the mutant, indicating that compound 1 is indeed R81 targeting GSHR. GSHR is a homodimeric protein that can reduce GSSG to GSH. Each monomer contains three domains: an NADPH binding domain, a FAD binding domain, and a dimerization domain. The crystal structure revealed that R81 is located at the entrance of the substrate NADPH binding pocket, adjacent to the disulfide bond active site. This example tests the effect of compound 1 labeling on the redox activity of GSHR, as shown in Figure 5. Figure 7 As shown in Figure D, it was found that compound 1 indeed inhibited the redox reactivity of GSHR. Moreover, this compound labeling was more effective than the R81A mutation, which may be due to the blocking of the enzymatic activity pocket of GSHR after labeling with compound 1.

[0200] In addition, according to the structure-activity analysis in this example, it was found that compound 1 can also target R97 of KAD1. Then, R97 of KAD1 was mutated, and the results were as expected, as shown in FIG. Figure 7As shown in Figure E, compound 1 is indeed targeting R97 of KAD1. KAD1 is an adenylate kinase that can transfer the phosphate on ATP to AMP to generate two molecules of ADP. It contains three domains: an ATP binding domain, an AMP binding domain, and an allosteric domain. The crystal structure revealed that R97 is located in the ATP binding pocket and may interact with the substrate phosphate in a salt bridge. So the effect of compound 1 labeling on KAD1 activity was tested, as shown in Figure 1. Figure 7 As shown in Figure F, it was found that compound 1 indeed inhibited the transfer of ATP phosphate, which may be due to the fact that compound 1 labeling blocked the interaction between KAD1 and phosphate.

[0201] Example 6

[0202] This embodiment provides a method for combining protein structure calculation and proteome analysis to find the key ligand arginine that mediates the interaction between proteins, and use inhibitor molecules to perturb this process. The process is as follows: Figure 8 As shown, the specific steps include:

[0203] In this example, the complex PDB of existing ligand arginine was first found from two protein complex databases HURI and hu.MAP v. 20. Since the solvent accessible surface area of ​​the monomeric ligand arginine decreases after forming a protein complex, the key ligand arginine mediating protein-protein interaction was screened by solvent accessible surface area calculation (SASA), and the solvent accessible surface area of ​​the ligand arginine when forming a protein complex was at least reduced by 50% compared with its monomer value.

[0204] Next, this example selected three pairs of complexes (CDK4-CCND1, CDK4-CCND3, PURA2-PURA1) according to the importance of the target and verified by co-immunoprecipitation whether the targeted small molecules could disrupt protein-protein interactions.

[0205] HEK293T cells were seeded in 6 cm culture dishes. After 24 hours, 5 μg of plasmids PURA2-Flag (plasmid PURA2-Flag was obtained by homologous recombination of PURA2 encoding gene fragment (nucleotide sequence as shown in GeneID: 159) and Flag tag into pLX304 expression vector), PURA1-V5 (plasmid PURA1-V5 was obtained by homologous recombination of PURA1 encoding gene fragment (nucleotide sequence as shown in GeneID: 122622) and V5 tag into pLX304 expression vector), and CDK4-Flag (plasmid CDK4-Flag was obtained by homologous recombination of CDK4 encoding gene into pLX304 expression vector) were transfected into HEK293T cells using 8 μL of Lipofectamine 8000 transfection reagent. The plasmid CCND1-V5 (plasmid CCND1-V5 was obtained by homologously recombining the CCND1 encoding gene fragment (nucleotide sequence as shown in GeneID: 1019) and the Flag tag into the pLX304 expression vector), CCND1-V5 (plasmid CCND1-V5 was obtained by homologously recombining the CCND1 encoding gene fragment (nucleotide sequence as shown in GeneID: 595) and the V5 tag into the pLX304 expression vector), and CCND3-V5 (plasmid CCND3-V5 was obtained by homologously recombining the CCND3 encoding gene fragment (nucleotide sequence as shown in GeneID: 896) and the V5 tag into the pLX304 expression vector) were transfected into cells. Approximately 24 hours after transfection, the cells were lysed by ultrasound with 50mM boric acid / HEPES buffer and quantified by BCA to 1.5mg / mL. Then, the protein with small molecule targeting arginine (Flag tag) was treated with small molecules (CP1, CP29) or DMSO (control group) at room temperature for 4 hours. The treated lysate was then incubated with the lysate expressing the V5-tagged protein at room temperature for 1 hour. Next, the sample was mixed with 5 μL of Flag magnetic beads and rotated overnight at 4°C. Finally, the beads were washed with 1 mL of boric acid / HEPES buffer, PBST (PBS containing 0.2% Tween 20), and PBS for 10 minutes each, all at room temperature. After washing, the beads were heated at 95°C for 10 minutes and immunoblotted using 4× Laemmli buffer, followed by anti-V5 antibody (purchased from CST) and anti-Flag antibody (purchased from CST).

[0206] like Figure 8 As shown, as expected, 500 μM small molecule ligands could significantly disrupt the CDK4-CCND1, CDK4-CCND3, and PURA2-PURA1 interactions.

[0207] In summary, some embodiments of the present invention conduct chemical proteomics research on arginine reactivity, which can obtain new highly active arginine sites, thereby realizing the functional research of unknown proteins; and through competitive screening of fragmented electrophilic small molecule libraries, arginine sites targeted by fragmented small molecules can be found, and new fragmented ligands or inhibitors can be found for undruggable proteins. Finally, by focusing on the regulatory activity of arginine sites in mediating protein-protein interactions, inhibitor molecules can be found to perturb this process.

[0208] Some embodiments of the present invention combine protein structure calculation and proteome analysis for the first time to find ligands that target protein-protein interactions. This discovery opens up new possibilities for drug development, especially in targeting protein-protein interactions that were previously considered undruggable.

[0209] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An application of a compound based on glyoxal in the preparation of a probe, characterized in that: The compound has a structure shown in Formula I: Wherein, R1 is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms; The substitutions are each independently substituted by at least one of halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, C1-C4 alkoxy, and phenoxy, wherein the number of substitutions ranges from a single substitution to a maximum number of substitutions; The probe has the application of any one of A1) to A4): A1) Detection or auxiliary detection of arginine; A2) preparing a product for detecting or assisting in detecting arginine; A3) screening or assisting in screening compounds that bind to arginine; A4) Preparation of products for screening or assisting screening of compounds that bind to arginine.

2. The use according to claim 1, characterized in that: R1 is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, naphthyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1 or 2 heteroatoms; Optionally, R1 is selected from methyl, ethyl, substituted or unsubstituted phenyl, naphthyl; Optionally, the substitutions are each independently substituted by at least one of F, Cl, Br, C1-C4 alkyl, C1-C4 alkyl substituted with F, C1-C4 alkoxy, and phenoxy, wherein the number of substitutions is mono- or di-substitution; Optionally, the substitutions are each independently substituted by at least one of F, Cl, Br, methyl, trifluoromethyl, methoxy, and phenoxy, wherein the number of substitutions is mono- to di-substitutions.

3. A probe, characterized in that: The probe is obtained by modifying the compound described in claim 1 or 2 with a functional group through a click reaction.

4. Use of the probe according to claim 3 in any one of A1) to A4), A1) Detection or auxiliary detection of arginine; A2) preparing a product for detecting or assisting in detecting arginine; A3) screening or assisting in screening compounds that bind to arginine; A4) Preparation of products for screening or assisting screening of compounds that bind to arginine.

5. A kit, characterized in that: The kit comprises the probe according to claim 3.

6. A method for detecting or assisting in detecting arginine using the probe according to claim 3, characterized in that: The following steps are involved: The probe is mixed with a protein sample and incubated to obtain a first system; the first system is mixed with a first reporter molecule to perform a first click reaction, and a proteomics analysis is performed on the reaction product to detect or assist in detecting arginine in the protein sample. Wherein, the first reporter molecule also contains a functional group that can undergo a click reaction with the probe.

7. A method for screening or assisting screening of compounds that bind to arginine using the probe according to claim 3, characterized in that: The following steps are involved: contacting the test compound with a protein sample containing arginine to obtain a second system, mixing the second system with the probe and incubating them to obtain a third system; mixing the third system with a second reporter molecule, performing a second click reaction, and analyzing the competitive binding effect of the test compound on arginine in the protein sample; Wherein, the second reporter molecule also contains a functional group that can undergo a click reaction with the probe.

8. The method according to claim 6 or 7, characterized in that: The first reporter molecule and the second reporter molecule each independently include at least one of biotin, desthiobiotin, and a fluorescent dye; Optionally, the first reporter molecule and the second reporter molecule each independently include a cleavable group; Optionally, in the first reporter molecule and the second reporter molecule, the functional group is linked to a cleavable group; Optionally, the cleavable group comprises a photocleavable group.

9. The method according to claim 8, characterized in that After the first click reaction or the second click reaction is completed, the protein is cleaved by protease to enrich the protein labeled by the first reporter molecule or the second reporter molecule, and then cleaved; Preferably, the cleavage is photocleavage; More preferably, the photolysis is performed under ultraviolet light conditions.

10. Use of glyoxal compounds in any one of 1) to 7): 1) preparing a reagent for binding arginine; 2) preparing a reagent for weakening or inhibiting protein-protein interaction, wherein: The protein-protein interaction domain includes arginine; 3) Preparation of inhibitors of CDK4-CCND3 interaction; 4) Preparation of CDK4-CCND1 interaction inhibitors; 5) preparing inhibitors of PURA2-PURA1 interaction; 6) Preparation of GSHR inhibitors; 7) preparing KAD1 inhibitors; Wherein, the glyoxal compound has a structure shown in formula (II): wherein R2 or R3 are each independently selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms; or R2 or R3 are connected to form a 6-9 membered spiro ring; The substitutions are each independently substituted by at least one of halogen, nitro, hydroxyl, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, and C1-C4 alkoxy, wherein the number of substitutions ranges from a single substitution to a maximum number of substitutions.

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