Application of a glyoxal-based compound in probe preparation

Abstract

This invention relates to the field of biotechnology, specifically to the application of glyoxal-based compounds in probe preparation. This invention develops glyoxal-based arginine-specific probes, utilizes proteomics analysis methods to perform a global analysis of the arginine reactivity in the proteome, constructs a fragmented electrophilic small molecule library with a glyoxal structure, and discovers new inhibitors for undrugable proteins through competitive proteomics. Finally, combining protein structure calculations and proteomics analysis, key ligand arginine molecule mediating protein-protein interactions is identified, and this process is perturbed using inhibitor molecules.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of a glyoxal-based compound in the preparation of probes. Background Technology

[0002] Arginine is an abundant amino acid (5.1%) that plays a crucial role in protein structure and function. It possesses a strongly basic guanidine group and is the second most abundant amino acid in protein-protein interaction hotspots. Notably, due to the negative charge of the phosphate group on nucleic acids, arginine is also a key site mediating protein-DNA or RNA interactions, playing a vital role in cellular processes such as RNA modification and cleavage. Arginine is also an important post-translational modification target, such as methylation and citrullination. Arginine methylation is primarily catalyzed by the protein arginine methyltransferase (PRMT) family. It mainly occurs on membrane receptors, histones, DNA damage proteins, and RNA-binding proteins, playing a crucial role in gene transcription, DNA damage repair, RNA cleavage, and cancer. Furthermore, arginine cation-π interactions play a significant role in regulating liquid-liquid phase separation (LLPS), and the methylation state of arginine can modulate the strength of these cation-π interactions. Hypomethylated arginine strongly promotes phase separation. Citrullination is a key post-translational modification (PTM) affecting protein structure and function. Catalyzed by human arginine deiminase, it converts positively charged arginine to neutral citrulline, leading to alterations in intramolecular and extramolecular interactions and protein conformational changes. Because citrullination significantly impacts protein structure and folding, some epitopes may change or new epitopes may form during protein citrullination. This, in turn, can trigger an immune response against the citrullinated host protein, potentially leading to autoimmune reactions, rheumatoid arthritis, and other conditions.

[0003] The reaction of intracellular dicarbonyl compounds with arginine was actually reported decades ago. Subsequently, 1,2-cyclohexanedione was commonly used to block arginine on trypsin. In addition, methylglyoxal and 2,3-butanedione have been used to modify arginine; however, these molecules simultaneously label other nucleophilic amino acids. In contrast, phenylglyoxal shows improved reaction rate and selectivity with arginine. Wager et al. used azide-modified phenylglyoxal to selectively label arginine on antibodies. Dong developed the arginine-lysine crosslinking agent KARGO and analyzed the interaction of the complex using crosslinking mass spectrometry. Therefore, designing arginine-specific chemical proteomics probes based on the structure of phenylglyoxal has a certain feasibility.

[0004] Ye Mingliang's research group recently developed a method for enriching dimethylated arginine based on steric hindrance, achieving global analysis of phase separation regulated by arginine dimethylation. Phenylacetaldehyde fragments preferentially react with citrulline (better than arginine) at acidic pH to form imidazolone ring structures. Thompson et al. used a biotin-modified phenylglyoxal probe to label citrullinated proteins under acidic conditions (pH = 2-3). Li Lingjun's research group reported a method for enriching and identifying citrullinated proteins using 2,3-butanedione and biotinylate tags, enabling a global study of the citrullinated proteome in mouse tissues.

[0005] In summary, research on the reactivity of arginine and its targetability to small molecules at the whole proteome level remains incomplete. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, some embodiments of this invention propose the application of glyoxal-based compounds in the preparation of probes.

[0007] Furthermore, some embodiments of the present invention utilize glyoxal-based arginine-specific probes and proteomics analysis methods to perform a global analysis of the arginine reactivity in the proteome.

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

[0009] In one aspect, this invention provides the application of glyoxal-based compounds in the preparation of probes, said compounds having the structure shown in Formula I (where R1 groups and H are respectively attached to the two carbonyl carbons in the glyoxal structure):

[0010]

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

[0012] Each of the substitutions is 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 monosubstituted to the maximum number of substitutions;

[0013] The probe has any one of the applications listed in A1) to A4):

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

[0015] A2) Prepare products for the detection or auxiliary detection of arginine;

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

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

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

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

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

[0021] According to some embodiments of the present invention, each of the substitutions is independently monosubstituted by at least one of methyl, trifluoromethyl, methoxy, and phenoxy, or each of the substitutions is 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- to 6-membered heteroaryl containing 1 or 2 heteroatoms.

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

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

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

[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 reagent kits, chips, or systems.

[0029] In another aspect, the present invention provides a probe obtained by modifying the functional groups of the above-mentioned glyoxal-based compound via a click reaction.

[0030] According to some embodiments of the present invention, the functional group of the click reaction is selected from alkyne group and 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-mentioned compound connected to a terminal alkyne group or a terminal azido 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-mentioned compound connected to a terminal alkyne group or a terminal azide group via one or two 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 application of the above-described probe in any one of A1) to A4).

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

[0037] A2) Prepare products for the detection or auxiliary detection of arginine;

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

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

[0040] According to some embodiments of the present invention, the product is selected from reagent kits, chips, or systems.

[0041] In another aspect, the present invention provides a kit comprising the probes described above.

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

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

[0044] A2) Prepare products for the detection or auxiliary detection of arginine;

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

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

[0047] According to some embodiments of the present invention, the product is selected from reagent kits, chips, or systems.

[0048] In another aspect, the present invention provides a method for detecting or assisting in the detection of arginine using the above-described 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 the reaction product is subjected to proteomics analysis to detect or assist in the detection of arginine in the protein sample.

[0050] The first reporter molecule also contains functional groups that can undergo a click reaction with the probe.

[0051] The principle of this method is as follows: The probe contains a glyoxal reactive group and a click-reaction functional group. Arginine in the protein, as a nucleophilic amino acid residue, contains a guanidinium group, which can react with the glyoxal group in the probe, thus forming a covalently bound complex between the protein and the probe. This complex is further reacted with a first reporter molecule, where the functional groups on the probe can undergo a click reaction with the functional groups on the first reporter molecule, thereby attaching a reporter group to the protein-probe complex 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 exhibits 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 carried out 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 reporter molecules well known in the art for enrichment purposes (e.g., biotin, dethiobiotin) or various reporter molecules for protein tracing 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, as a detectable marker, may include at least one of biotin and carboxytetramethylrhodamine. In some embodiments of the present invention, biotin-labeled protein targets can be enriched by the binding of streptavidin to biotin, facilitating 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. Therefore, the functional group can be selected from alkyne or azide groups. 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 azide group; and when the functional group contained in the probe is an azide 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-butyltrichloroacetylimide, 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 further includes a cleavable group, which includes, but is not limited to, a photocleavable group (PC), an acid cleavable group (DADPS), and an enzyme cleavable group (TEV), and the cleavable group is linked to a 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 linked by 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 digested with a protease to enrich the protein of the first reporter molecular marker, and then cleaved.

[0062] According to some preferred embodiments of the present invention, the enzymatic digestion is performed using trypsin.

[0063] According to some preferred embodiments of the present invention, the pyrolysis is photopyrolysis.

[0064] According to some preferred embodiments of the present invention, the photolysis is carried out under ultraviolet light conditions, and the time of photolysis is not particularly limited; for example, the photolysis is carried out at 365 nm for 0.5 to 2 hours to release peptides.

[0065] According to some embodiments of the present invention, the detection or auxiliary detection of arginine in the protein sample includes detecting the presence or absence of arginine in the protein sample, and also includes assessing 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 yield at different probe concentrations. For example, the reactivity of arginine in the proteome can be quantified by comparing the ratio of primary peptide ionic strength (Ratio H / L).

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

[0068] The test compound is contacted with a protein sample containing arginine to obtain a second system. The second system is then mixed with the probe and incubated to obtain a third system. The third system is then mixed with a second reporter molecule to perform a second click reaction, and the competitive binding effect of the test compound on arginine in the protein sample is analyzed.

[0069] The second reporter molecule also contains functional groups that can undergo a click reaction with the probe.

[0070] According to some embodiments of the present invention, the contact is carried out at 20–35°C for 2–6 hours.

[0071] According to some embodiments of the present invention, the quantification standard of the competitive binding effect is as follows: arginine reacting with the test compound will competitively inhibit the labeling of the probe, resulting in a significant reduction in the labeling degree. Compared with the DMSO group, the primary peptide ion strength of the test compound pretreatment group is significantly reduced. This 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, incubation conditions, second reporter molecule, reaction conditions for the second click reaction, and steps after the second click reaction are the same as or refer to the methods for detecting or assisting in the detection of arginine described above, and will not be repeated here.

[0073] Accordingly, using the screening or auxiliary screening methods described above, some embodiments of the present invention have discovered new inhibitors for proteins that cannot be drugged by competitively binding to arginine in protein samples through fragmented electrophilic small molecule libraries.

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

[0075] In another aspect, the present invention provides the use of glyoxal compounds in the preparation of reagents that weaken or inhibit protein-protein interactions, wherein the protein-protein interaction domain includes arginine.

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

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

[0078] The interaction between CDK4-CCND3 and / or CDK4-CCND1 plays a crucial role in the development and progression of various diseases, such as B-cell acute lymphoblastic leukemia, neurological dysfunction, and especially cancer. Therefore, the glyoxal compounds described herein possess therapeutic potential for B-cell acute lymphoblastic leukemia, neurological dysfunction, and especially cancer.

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

[0080] The PURA2-PURA1 interaction is associated with the following diseases or conditions: PURA2 and PURA1 genes play important roles in cardiac energy metabolism and function, and their interaction with the cardiac transcription factor GATA4 has a significant impact on cardiac gene expression and function. Therefore, the glyoxal compounds described herein have therapeutic potential for diseases associated with disorders of cardiac gene expression and function.

[0081] In another aspect, the present invention provides the 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 R81 amino acid of GSHR, thereby inhibiting the redox activity of GSHR.

[0083] In another aspect, the present invention provides the 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 the structure shown in formula (II):

[0086]

[0087] R2 or R3 is independently selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted biphenyl, substituted or unsubstituted 5-6 heteroaryl containing 1, 2 or 3 heteroatoms; or R2 or R3 are linked to form a 6-9 spirocyclic ring.

[0088] Each of the substitutions is 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 monosubstituted to the maximum number of substitutions.

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

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

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

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

[0093] According to some embodiments of the present invention, the glyoxal compounds are selected from the following structural formulas:

[0094]

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

[0096] An arginine-specific probe based on glyoxal was developed, and the global reactivity of arginine in the human proteome was analyzed using proteomics methods. Furthermore, new inhibitors for undrugifiable proteins were discovered through competitive screening of fragmented electrophilic small molecule libraries. Finally, the inhibitor molecules were used to perturb protein-protein interactions.

[0097] Many terms will be used in this specification and claims, and these terms shall have the following definitions:

[0098] The term "arginine" includes arginine ( Arginine or its derivatives, wherein “derivatives” are defined herein as chemical moieties covalently attached to arginine (other than the guanidinium group on the side chain), such as acids, esters, amides, alcohols, alcohol derivatives, aldehydes, amines, salts, hydrates, glycosides, isomers, and enantiomers. It is understood that arginine or its derivatives contain a guanidinium group.

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

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

[0101] The term "halogen-substituted C1-C4 alkyl" refers to a C1-C4 alkyl group substituted with one or more halogens, 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 "heteroatoms" refers to atoms such as O, S, N, Se, Si, or Ge.

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

[0104] The term “substituted or unsubstituted” means that one or more hydrogen atoms are replaced by other atoms or functional groups (i.e., substituents), and unless otherwise defined, also includes the replacement of one or more hydrogen atoms by groups formed by the linkage of two or more of the substituents described above.

[0105] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

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

[0107] Figure 1 This is a flowchart of the process of using probe 1 to identify arginine sites in the human proteome.

[0108] Figure 2 This is a flowchart and results diagram of probe screening. A: Probe optimization workflow combining LC-MS / MS and open search; B: Analysis of the number of arginine-modified peptide match profiles (PSM) in four different groups with PG as an internal standard.

[0109] Figure 3The figure shows the results of the identification depth and selectivity studies for probe 1 and probe 2. In the figure, A: the modification distribution of probes 1 and 2 is analyzed, considering the case where the peptide match spectrum number (PSM) value is greater than 80; B: the selectivity of probes 1 and 2 for arginine is evaluated using an open library search method; C: the number of modification sites is determined by using a closed library search method with the expected modification offset quality of the two probes; D: the selectivity of probes 1 and 2 is determined by using the offset quality described in A through an offset library search method.

[0110] Figure 4 This is a graph showing the results of mass spectrometry sample processing workflow optimization and identification of arginine sites in the human proteome. A: Identification and analysis of arginine-labeled sites in membrane proteins and soluble protein components using probe 1, including two biological replicates. B: Comparison of an alternative workflow (where trypsin digestion precedes enrichment) with the traditional method (enrichment precedes digestion) for identifying arginine-labeled sites, with two biological replicates. C: Schematic diagram of the optimized workflow, detailing the pre-treatment of trypsin digestion before enrichment using probe 1. D: Good reproducibility of labeled arginine sites in the 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: Heatmap showing the complementarity of labeled arginine sites in different cell lines.

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

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

[0113] Figure 7 This is a graph showing the results of ligand targeting studies in the human proteome. A: A schematic diagram illustrating the competitive DIA-ABPP workflow used to identify arginine binding to glyoxal compounds. B: The ratio of ligand-dependent protein (left) to ligand-dependent arginine (right) compared to quantitative protein and arginine. C: Western blot 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 the R81A mutation on the redox-dependent enzyme activity of GSHR. E: Western blot 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 graph showing the results of a study targeting key arginine residues to interfere with protein-protein interactions. A: A schematic diagram illustrating the change in solvent-accessible surface area (SASA) of ligand-dependent arginine residues during complex formation. B: A heatmap showing ligand-dependent arginine residues that may mediate protein-protein interactions. These arginine residues showed a SASA value reduction of at least 50% after binary protein complex formation. CH: A representative co-immunoprecipitation assay confirms the disruption of selected protein-protein interactions by fragmented small molecules. The bar chart shows the protein abundance quantification from Western blotting analysis, with data presented as the mean ± standard deviation of three biological replicates. Detailed Implementation

[0115] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

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

[0117] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

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

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

[0120] Some embodiments of the present invention utilize proteomics analysis methods to globally analyze the reactivity of arginine in the human proteome by developing an arginine-specific probe 1 based on phenylglyoxal. Furthermore, some embodiments of the present invention discover new inhibitors for undrugizable 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 glyoxal derivatives and adding an azide tag to obtain arginine-specific probe 1; optimizing reaction conditions and proteomics research workflows, mixing the azide-tagged arginine-specific probe 1 with protein lysis buffer, then conducting a click reaction with a biotin-tagged alkyne reagent, and finally performing proteomics analysis to obtain arginine sites with different reactivity; purchasing commercially available fragmented electrophilic small molecule libraries with glyoxal structures, and discovering new inhibitors for undrug-resistant proteins through competitive proteomics; finally, combining protein structure calculations and proteomics analysis to find key ligand arginine that mediates protein-protein interactions, and perturbing this process using inhibitor molecules.

[0122] Example 1

[0123] This embodiment develops an arginine-specific probe 1 based on phenylglyoxal (compound B3 with an azide tag), and its screening process includes the following steps:

[0124] A focused molecular library of 13 molecules was constructed using glyoxal derivatives purchased from Bid Pharmaceuticals.

[0125]

[0126] Thirteen small molecules (PG, A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3) were dissolved in DMSO to prepare 200 mM stock solutions. The 13 small molecules were grouped into four groups, with PG serving as an internal standard in each group. 10 μL of each of the three small molecules (PG, A1, A2, A3) was mixed to form Group A (50 mM concentration of each molecule). Similarly, 10 μL of each of the three small molecules (PG, B1, B2, B3) was mixed to form Group B (50 mM), 10 μL of each of the three small molecules (PG, C1, C2, C3) was mixed to form Group C (50 mM), and 10 μL of each of the three small molecules (PG, D1, D2, D3) was mixed to form Group D (50 mM).

[0127] Six proteins (RNase A, catalase, Lysozyme, lactoferrin, aldolase, and β-amylase) were dissolved in PBS to prepare stock solutions of 2 mg / mL each. 7.14 μL of each protein stock solution was added to a 100 μL PBS system to prepare a 1 mg / mL protein mixture.

[0128] Then, 2 μL of a 50 mM mixture of small molecules A, B, C, and D was added to 100 μL of a protein mixture and reacted for 2 hours. The resulting system was mixed with 5 times its volume of pre-cooled acetone to precipitate, and then reconstituted with 60 μL of 25 mM ABC (ammonium carbamate) containing 6 M urea. Next, the resulting system was reduced with 10 mM DTT (dithiothreitol) at 55 °C for 40 minutes and cooled to room temperature. 15 mM IAA (iodoacetamide) was added, and the mixture was alkylated at room temperature in the dark for 30 minutes using a rotary mixer. The IAA reaction was quenched by adding 5 mM DTT solution (termination was achieved by 10 minutes at room temperature using a rotary mixer). Next, 300 μL of 25 mM ABC was added to dilute the Urea concentration in the resulting system to 1 M, and 1 mM CaCl2 (calcium ions are used to increase trypsin activity) was added, along with 1:100 trypsin (i.e., 1 μg trypsin added to 100 μg of protease). The system was then incubated overnight at 37°C with shaking (1050 rpm, approximately 12 hours). Finally, the resulting system was placed on ice, and 20% (v / v) formic acid was slowly added to adjust the pH to 2-3 (monitored with pH paper) to quench the trypsin digestion reaction. After desalting using a 96-well desalting plate (capable of withstanding 20 μg peptide in a 200 μL system), 0.1% (v / v) formic acid was added to reconstitute the solution, and the sample was injected for LC-MS / MS analysis. Mass spectrometry data were searched using a database.

[0129] Open search results such as Figure 1As shown, B3 and D1 have the highest number of arginine-modified peptide match patterns (PSMs), which are 3.0 times and 2.2 times higher than the PG internal standard, respectively. Therefore, B3 and D1 were chosen to be tagged with an azide to synthesize probe 1 and probe 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). The mixture was heated to 100 °C with stirring and reacted at this temperature for 16 hours. After cooling to room temperature, ethyl acetate (100 mL) and water (300 mL) were added, and the mixture was stirred to separate the layers. The aqueous phase was extracted with ethyl acetate (100 mL), and the organic phases were combined. The mixture was 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 give the target product (6.60 g, 40.5%) as a light brown solid.

[0135] Synthesis step 2 of probe 1: Synthesis of 1-(4-(4-(bromomethyl)phenoxy)phenyl)ethane-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 benzoyl peroxide (650.0 mg, 10% wt). The mixture was heated to reflux and stirred under reflux for 16 hours. After cooling to room temperature, water (50 mL) was added, and the mixture was stirred to separate the layers. The aqueous phase was extracted with dichloromethane (50 mL), and the organic phases were combined. The organic phase was washed with saturated brine (40 mL), and the separated organic phase was concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to give the target product (5.52 g, 62.9%) as an off-white solid.

[0138] Synthesis step 3 of probe 1: Synthesis of 1-(4-(4-(azidomethyl)phenoxy)phenyl)ethane-1-one:

[0139]

[0140] 1-(4-(4-(bromomethyl)phenoxy)phenyl)ethane-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 portions. After the addition was complete, the mixture was stirred at room temperature for 3 hours. Water (200 mL) and ethyl acetate (55 mL) were added, and the mixture was stirred to separate the layers. The aqueous phase was extracted with ethyl acetate (35 mL), and the organic phases were combined and washed with saturated brine (35 mL). The mixture was concentrated to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to give the target product (4.10 g, 85.1%) in an oily state.

[0141] Step 4 of the synthesis of probe 1: 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), and the mixture was cooled to 0 °C under nitrogen protection. The temperature was maintained below 5 °C, and 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 and reacted for 3 hours. Water (60 mL) was added, and the mixture was stirred to separate the layers. The 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 give a light brown oily target product (5.40 g, N / A). The crude product was used directly in the next reaction.

[0144] Synthesis step 5 of probe 1: Synthesis of 1-(4-(4-(azidomethyl)phenoxy)phenyl)-2-((trimethylsilyl)oxy)ethane-1-one:

[0145]

[0146] (1-(4-(4-(azidomethyl)phenoxy)phenyl)vinyl)oxy)trimethylsilane (5.40 g, 15.339 mmol, 1.0 eq, crude product from the previous step, in 100% yield) was dissolved in anhydrous dichloromethane (29 mL, 5V). Under nitrogen protection, a dichloroperoxybenzoic acid solution (3.97 g, 23.009 mmol, 1.5 eq) in 58 mL of dichloromethane was added dropwise. After addition, the mixture was transferred to room temperature and stirred for 5 hours. The reaction was quenched by adding saturated sodium bicarbonate and saturated sodium thiosulfate solutions, respectively. The mixture was stirred to separate the phases. The aqueous phase was extracted with dichloromethane (30 mL). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the oily target product (5.70 g, N / A). The crude product was used directly in the next reaction step.

[0147] Synthesis step 6 of probe 1: 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, in 100% yield) was dissolved in anhydrous tetrahydrofuran (57 mL, 10 V), and a 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (18.41 mL, 18.407 mmol, 1.2 eq) was added dropwise. After the addition was complete, 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 to separate the layers, and the 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 subjected to silica gel column chromatography (PE:EA = 5:1) to give the target product (1.06 g, 24.5%) as a pale yellow solid.

[0150] Synthesis step 7 of probe 1: 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). The mixture was heated to reflux and stirred for two hours. After cooling, the solution was filtered through diatomaceous earth. The filtrate was concentrated to dryness, and the crude product was subjected to silica gel column chromatography (PE:EA = 2:1) to give the target product as a 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-methylnaphth-2-yl)ethane-1-one

[0156]

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

[0158] Step 2 of the synthesis of probe 2: Synthesis of 1-(6-(bromomethyl)naphth-2-yl)ethane-1-one

[0159]

[0160] 1-(6-methylnaphthyl-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 benzoyl peroxide (520.0 mg, 10% wt). The mixture was heated to reflux and stirred under reflux for 16 hours. After cooling to room temperature, water (52 mL) was added, and the mixture was stirred to separate the layers. The aqueous phase was extracted with dichloromethane (35 mL), and the organic phases were combined and washed with saturated brine (35 mL). The separated organic phase was concentrated to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to give the target product (4.86 g, 65.5%) as an off-white solid.

[0161] Step 3 of the synthesis of probe 2: Synthesis of 1-(6-(azidomethyl)naphth-2-yl)ethane-1-one

[0162]

[0163] 1-(6-(bromomethyl)naphthyl-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 portions. After the addition was complete, the mixture was stirred at room temperature for 3 hours. Water (196 mL) and ethyl acetate (50 mL) were added, and the mixture was stirred to separate the layers. The aqueous phase was extracted with ethyl acetate (30 mL), and the organic phases were combined and washed with saturated brine (30 mL). The mixture was concentrated to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give the target product (3.42 g, 83.2%) in an oily state.

[0164] Step 4 of the synthesis of probe 2: Synthesis of (1-(6-(azidomethyl)naphth-2-yl)vinyl)oxy)trimethylsilane

[0165] 1-(6-(azidomethyl)naphth-2-yl)ethane-1-one (3.40 g, 15.094 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (51 mL, 15 V), and the mixture was cooled to 0 °C under nitrogen protection. The temperature was maintained below 5 °C, and 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 and reacted for 3 hours. Water (51 mL) was added, and the mixture was stirred to separate the layers. The 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 give a light brown oily target product (4.70 g, N / A). The crude product was used directly in the next reaction.

[0166] Step 5 of the synthesis of probe 2: Synthesis of 1-(6-(azidomethyl)naphth-2-yl)-2-((trimethylsilyl)oxy)ethane-1-one

[0167]

[0168] (1-(6-(azidomethyl)naphth-2-yl)vinyl)oxy)trimethylsilane (4.70 g, 15.094 mmol, 1.0 eq, crude product from the previous step, in 100% yield) was dissolved in anhydrous dichloromethane (24 mL, 5 V). Under nitrogen protection, a dichloroperoxybenzoic acid solution (3.91 g, 22.641 mmol, 1.5 eq) in dichloromethane (47 mL, 10 V) was added dropwise. After addition, the mixture was transferred to room temperature and stirred for 5 hours. The reaction was quenched by adding saturated sodium bicarbonate and saturated sodium thiosulfate solutions, respectively. The mixture was stirred to separate the phases. The aqueous phase was extracted with dichloromethane (30 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to dryness to obtain the oily target product (4.98 g, N / A), which was directly used in the next reaction.

[0169] Step 6 of the synthesis of probe 2: Synthesis of 1-(6-(azidomethyl)naphth-2-yl)-2-hydroxyethane-1-one

[0170]

[0171] 1-(6-(azidomethyl)naphth-2-yl)-2-((trimethylsilyl)oxy)ethane-1-one (4.98 g, 15.094 mmol, 1.0 eq, crude product from the previous step, based on 100% yield) was dissolved in anhydrous tetrahydrofuran (50 mL, 10 V), and a 1 mol / L tetrabutylammonium fluoride tetrahydrofuran solution (18.2 mL, 18.113 mmol, 1.2 eq) was added dropwise. After the addition was complete, 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 to separate the layers, and the 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 subjected to silica gel column chromatography (PE:EA = 5:1) to give the target product (746.5 mg, 20.5%) as a pale yellow solid.

[0172] Step 7 of the synthesis of probe 2: Synthesis of 2-(6-(azidomethyl)naphth-2-yl)-2-oxoacetaldehyde

[0173]

[0174] 1-(6-(azidomethyl)naphth-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). The mixture was heated to reflux and stirred for two hours. After cooling to room temperature, the mixture was filtered through diatomaceous earth. The filtrate was concentrated to dryness, and the crude product was subjected to silica gel column chromatography (PE:EA = 2:1) to give the target 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 embodiment uses mass spectrometry to compare the identification depth and selectivity of probe 1 and probe 2 at the whole proteome level. Open library search results are as follows: Figure 2As shown, the results indicate that probe 1 has a higher selectivity for labeling arginine than probe 2, and probe 1 can identify about 1600 sites, while probe 2 can only identify about 600 sites. Therefore, probe 1 was selected as the arginine-specific probe for subsequent proteomics studies.

[0176] Example 2

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

[0178] HeLa cells were seeded in 15 cm culture dishes. When the cell density reached 90%, the cells were washed twice with pre-chilled PBS, scraped, and resuspended in 50 mM borate / HEPES buffer containing EDTA-free protease inhibitors. The cells were sonicated for 1 minute, centrifuged at 14,000 g for 10 minutes at 4°C to remove debris, and the BCA lysis buffer was quantitatively adjusted to 1.5 mg / mL. 4 mL of the lysis buffer was incubated with 1 mM probe 1 at room temperature for 4 hours. Then, the cells were reacted with photolyzable alkyne-biotin. Click reaction was performed using CAS No.: 1869922-24-6 (room temperature, 1 hour, 0.1 mM alkyne-biotin, 1 mM trichloroethyl phosphate (TCEP) reducing agent, 0.1 mM tert-butyltrichloroacetylimide (TBTA) ligand, and 1 mM CuSO4). After the click reaction, the reaction solution and the precipitation solution (methanol and chloroform mixture) were mixed at a ratio of 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, and 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 buffer containing 10 mM dithiothreitol (DTT) was added, and the mixture was reduced at 55 °C for 40 minutes. Finally, the precipitate was alkylated with 15 mM fresh iodoacetamide (IAA) in the dark at room temperature for 30 minutes. Next, the resulting system and the precipitate solution were mixed at a ratio of 5 mL pre-cooled acetone to 1 mL of the resulting system, and centrifuged at 4,500 g for 10 minutes at room temperature. The supernatant was discarded, and the precipitate was washed with 1 mL of acetone and reconstituted with 2 mL of 25 mM ammonium carbamate (ABC) containing 1 M urea. 1 mM CaCl2 and 100 μg of trypsin were added, and the mixture was shaken overnight at 37 °C for digestion. Then, the resulting system was diluted with 2 mL of PBS and incubated with 400 μL of streptavidin beads at room temperature for 4 hours. The beads were washed three times with 5 mL of PBS containing 0.2% SDS, three times with 5 mL of PBS containing 1 M urea, and three times with 5 mL of PBS. Finally, after washing three times with 1 mL of distilled water, the beads were resuspended in 600 μL of 70% methanol-water solution in a 24-well plate, placed on ice, and irradiated with 365 nm light for 60 minutes to release the peptides. The supernatant from the 24-well plates was collected in 1.5 mL low-binding EP tubes. The magnetic beads in the 24-well plates were washed twice with 400 μL of 70% methanol-water solution. The combined samples were dried in a SpeedVac vacuum concentrator. For LC-MS / MS analysis, the samples were redissolved in 0.1% (v / v) formic acid-water solution and analyzed using a QE Plus mass spectrometer equipped with a nano-spray ionization (nano-ESI) source and Xcalibur 4.3 software.

[0179] Example 3

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

[0181] First, this embodiment compares the fractionation and non-fractionation of membrane plasma components in the site identification process. The fractionation process is as follows: HeLa cells are scraped and resuspended in 50mM boric acid / HEPES buffer, sonicated for 1 minute, and the cell membrane and plasma components are separated using an ultracentrifuge at 100,000g, 4°C for 45 minutes. The supernatant is collected into a new EP tube. 400μL of 50mM boric acid / HEPES solution containing 0.5% Triton X-100 is added to the precipitate, and the mixture is sonicated again for 1-2 seconds to dissolve. After BCA quantification and adjustment to the same concentration, the steps in Example 2 are followed: the membrane and plasma components are treated using probe 1, followed by click reaction to ligand-biotin, trypsin digestion, streptavidin magnetic bead enrichment, optical cleavage, and LC-MS / MS identification.

[0182] Next, (without grading) different enrichment methods in the site identification process were compared. The enrichment-then-enzymatic digestion process is as follows: Referring to the steps in Example 2: HeLa lysis buffer is treated with probe 1, alkynyl-biotin is linked by click reaction, streptavidin magnetic beads are used for enrichment, trypsin digestion is performed, optical cleavage is performed, and LC-MS / MS is used for identification. The enzymatic digestion-then-enrichment process is as follows: Referring to the steps in Example 2: HeLa lysis buffer is treated with probe 1, alkynyl-biotin is linked by click reaction, trypsin digestion is performed, streptavidin magnetic beads are used for enrichment, optical cleavage is performed, and LC-MS / MS is used for identification.

[0183] The results are as follows Figure 4 As shown, the study found that the number of sites identified by fractionated and non-fractionated membrane slurry components was comparable. Furthermore, a comparison of different enrichment methods during sample processing revealed that enzymatic digestion followed by enrichment resulted in the identification of more arginine sites: using probe 1 to treat HeLa lysate, followed by click reaction to ligand-biotin, trypsin digestion, streptavidin magnetic bead enrichment, and optical cleavage, approximately 2500 sites were identified by LC-MS / MS, about three times more than the initial PG probe. The reproducibility of the label was confirmed through multiple biological replication experiments.

[0184] Finally, following the steps outlined in Example 3, HeLa cells were replaced with Ramos cells, MDA-MB-231 cells, or A549 cells to identify and label various cell lines. Consistent with existing literature reports, the proteomic compositions of the MDA-MB-231 cell line and the Ramos cell line are complementary. Therefore, a total of 4606 arginine sites were identified in different cell lines, verifying the complementarity between MDA-MB-231 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 labeling on the samples treated with two probe concentrations respectively.

[0188] Referring to the steps in Reference 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 at 37 °C overnight, 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 � 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 ion intensity of the first-level peptide segment "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), RH:L > 4 (low reactivity) respectively.

[0189] Next, the reactivity of the arginine site was verified using in-gel fluorescence assays. 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. Approximately 24 hours after transfection, cells were lysed by sonication with 50 mM borate / HEPES buffer, and BCA concentration was quantified to 1.5 mg / mL. Probe 1 at a final concentration of 200 μM was added to 300 μL of lysis buffer and incubated at room temperature for 4 hours. Then, 30 μL of pre-washed Flag beads were added, and enrichment was carried out overnight at 4°C. 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, followed by competition at room temperature for 1 hour. The Flag beads were then adsorbed onto a magnetic rack, and the supernatant was collected. Finally, a 30 μL click reaction cocktail was prepared: TCEP (50 mM, final concentration 1 mM); TBTA ligand (1.7 mM, final concentration 100 μM); CuSO4 (50 mM, final concentration 1 mM); and TAMRA-Alkyne (carboxytetramethylrhodamine (TAMRA) alkyne, 10 mM, final concentration 100 μM). The mixture was incubated at room temperature for 1 hour. Then, 10 μL of 4× Loading buffer was added, and the mixture was boiled for 10 minutes before SDS-PAGE analysis.

[0190] This embodiment utilizes an arginine-specific probe 1 for proteomics analysis, performing a global analysis of arginine reactivity in the human proteome. The process involved labeling samples with high and low concentrations of probe 1, click reaction, trypsin digestion, and streptavidin magnetic bead enrichment. Then, isotope-labeled dimethylation modifications were introduced into the high and low concentration samples, followed by mixing and optical cleavage. Finally, the reactivity of arginine was identified by LC-MS / MS. Both high and low concentrations of the probe preferentially react with highly reactive arginine. Therefore, the reactivity of arginine in the entire proteome can be quantified by comparing the ratio of primary peptide ionic strength (Ratio H / L). Among all identified arginine sites, the vast majority showed probe concentration-dependent labeling, indicating low activity, while only a small subset of identified arginine sites exhibited high reactivity.

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

[0192] Furthermore, this embodiment utilizes parallel reaction monitoring (PRM) targeted mass spectrometry to validate proteins with low-reactivity arginine, including R115 of FKBP2 (GeneID:2286), R29 of KT3K (GeneID:79672), R48 of PARK7 (GeneID:11315), and R37 of CAPZA1 (GeneID:829). In proteins with mutations in several reactive arginines, the change in labeling of low-reactivity arginine is negligible, while in proteins with mutations in only a single reactive arginine, regardless of whether the arginine is low-reactivity or moderate-reactivity, labeling is blocked.

[0193] Example 5

[0194] This implementation provides a method for globally analyzing the targeting of arginine in the human proteome through competitive screening of fragmented electrophilic small molecule libraries, thereby discovering new inhibitors for undrugifiable proteins.

[0195] In this embodiment, 32 electrophilic fragment molecules targeting arginine were designed based on common pharmacophore structures in drug molecules, and then the small molecule targeting of arginine was evaluated using a "competitive proteomics" approach. Specifically, a glyoxal molecule library with different substituents was first purchased. The glyoxal molecule library was divided into three chemical types: aryl-substituted, alkyl-substituted, and monosubstituted.

[0196]

[0197] Next, the chemical properties of the small molecule library were investigated. It was found that most molecules conformed to the five principles of drug-like compounds and the three principles of lead compounds (e.g., ...). Figure 6 (As shown in the figure). Finally, the reaction specificity of the molecular library was studied. According to the literature, glyoxal compounds mainly react with the side chains of arginine, cysteine, lysine, and histidine. Therefore, the amino and carboxyl terms of the four amino acids were protected to obtain model amino acids, which were then reacted with molecules in the molecular library.

[0198] The method for global analysis of arginine targeting in the human proteome in this embodiment is performed following the steps in Example 2, such as... Figure 7 As shown, the proteome was pretreated with fragmented molecules or DMSO at room temperature for 4 hours, followed by incubation with probe 1 at room temperature for 4 hours, click reaction at room temperature for 1 hour, trypsin digestion at 37°C overnight, streptavidin magnetic bead enrichment at room temperature for 4 hours, and optical cleavage on ice for 1 hour. Finally, the ligand targeting of arginine was analyzed by DIA-based LC-MS / MS. Figure 7 As shown, 526 ligand-targetable proteins and 687 ligand-targetable arginine residues were identified. Arginine residues reacting with fragmented molecules competitively inhibit probe labeling, resulting in a significant reduction in labeling intensity (>75%). Compared to the DMSO control group, the proportion of primary peptide ionic strength was significantly lower. This degree of inhibition was reflected by the "ligand targeting ratio, RDMSO / CP," with an effective ligand defined as an RDMSO / CP ratio ≥ 4. Figure 6 As shown, LC-MS results indicate that none of the molecules react with lysine K or histidine H, while a small number react with cysteine ​​C to form unstable hemiacetals.

[0199] Next, in this embodiment, a heatmap was plotted for each identified arginine that can be targeted by the ligand, along with each ligand, and a structure-activity relationship analysis was performed. It was found that compound 1 (CP1) targets R81 of GSHR. Then, GSHR's R81 was mutated, and the results were as expected. Figure 7 As shown in Figure C, compound 1 can block the labeling of wild-type WT, while the labeling of mutant R81A is weakened, and compound 1 has no effect on the labeling of mutants, indicating that compound 1 is indeed the R81 that targets GSHR. GSHR is a homodimeric protein that can reduce GSSG to GSH. Each monomer contains three domains: an NADPH-binding domain, an FAD-binding domain, and a dimerization domain. Crystal structure analysis revealed that R81 is located at the entrance of the substrate NADPH binding pocket, adjacent to the disulfide active site. This example tested the effect of compound 1 labeling on the redox activity of GSHR, as shown in... Figure 7 As shown in Figure D, compound 1 indeed inhibited the redox reactivity of GSHR. Moreover, this compound labeling was more effective than the R81A mutation, possibly because compound 1 labeling blocked the enzyme activity pocket of GSHR.

[0200] Furthermore, based on structure-activity analysis, this embodiment found that compound 1 can also target R97 of KAD1. Subsequently, a mutation was performed on R97 of KAD1, and the results were as expected. Figure 7As shown in Figure E, compound 1 is indeed R97 targeting KAD1. KAD1 is an adenosine kinase that transfers phosphate from 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. Crystal structure analysis revealed that R97 is located in the ATP-binding pocket, potentially interacting with the substrate phosphate via a salt bridge. Therefore, the effect of compound 1 labeling on KAD1 activity was tested, as shown in... Figure 7 As shown in Figure F, compound 1 was found to inhibit the transfer of ATP phosphate, which may be due to the fact that compound 1 labels block the interaction between KAD1 and phosphate.

[0201] Example 6

[0202] This implementation example provides a method that combines protein structure calculations and proteomics analysis to identify key arginine ligands that mediate protein-protein interactions, and then uses inhibitory molecules to perturb this process. The procedure is as follows: Figure 8 As shown, the specific steps include:

[0203] This embodiment first identifies existing protein complex PDBs containing liganded arginine from two protein complex databases, HURI and hu.MAP v.20. Based on the fact that the solvent-accessible surface area of ​​monomeric liganded arginine decreases after protein complex formation, key liganded arginines mediating protein-protein interactions are screened using Solvent Accessible Surface Area (SASA) calculations. These liganded arginines exhibit a solvent-accessible surface area at least 50% lower than that of their monomers when forming the protein complex.

[0204] Next, based on the importance of the target, this embodiment selected three pairs of complexes (CDK4-CCND1, CDK4-CCND3, PURA2-PURA1) to verify whether the targeted small molecules could disrupt protein-protein interactions through immunoprecipitation.

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

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

[0207] In summary, some embodiments of this invention have conducted chemical proteomics studies on arginine reactivity, which can reveal new highly active arginine sites, thereby enabling functional studies of unknown proteins. Furthermore, through competitive screening of fragmented electrophilic small molecule libraries, arginine sites targeted by fragmented small molecules can be discovered, leading to the discovery of new fragmented ligands or inhibitors for undrugable proteins. Finally, by focusing on the regulatory activity of arginine sites in mediating protein-protein interactions, inhibitory molecules can be sought to perturb this process.

[0208] Some embodiments of this invention combine protein structure calculations and proteomics analysis for the first time to identify ligands that target protein-protein interactions. This discovery opens up new possibilities for drug development, particularly in targeting protein-protein interactions previously considered untreatable.

[0209] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a glyoxal-based compound for the preparation of a probe, characterized in that, The compound is selected from: 、 ； The probe has any one of the applications listed in A1) to A4): A1) Detection or auxiliary detection of arginine; A2) Prepare products for the detection or auxiliary detection of arginine; A3) Screening or assisting in the screening of compounds that bind to arginine; A4) Prepare products for screening or assisting in the screening of compounds that bind to arginine.

2. A probe, characterized in that, The probe is obtained by modifying the functional groups of the compound described in claim 1 via a click reaction.

3. The application of the probe as described in claim 2 in any of A1) to A4), A1) Detection or auxiliary detection of arginine; A2) Prepare products for the detection or auxiliary detection of arginine; A3) Screening or assisting in the screening of compounds that bind to arginine; A4) Prepare products for screening or assisting in the screening of compounds that bind to arginine.

4. A kit characterized in that, The kit includes the probe as described in claim 2.

5. A method of detecting or aiding in the detection of arginine using the probe of claim 2, wherein, Includes the following steps: The probe is mixed with a protein sample and incubated to obtain a first system; the first system is then mixed with a first reporter molecule to perform a first click reaction; the reaction product is then subjected to proteomics analysis to detect or assist in the detection of arginine in the protein sample. The first reporter molecule also contains functional groups that can undergo a click reaction with the probe.

6. A method of screening or aiding in the screening of compounds that bind arginine using the probe of claim 2, wherein, Includes the following steps: The test compound is contacted with a protein sample containing arginine to obtain a second system. The second system is then mixed with the probe and incubated to obtain a third system. The third system is then mixed with a second reporter molecule to perform a second click reaction, and the competitive binding effect of the test compound on arginine in the protein sample is analyzed. The second reporter molecule also contains functional groups that can undergo a click reaction with the probe.

7. The method of claim 5, wherein, The first reporter molecule includes at least one of biotin, desulfobiotin, and fluorescent dye.

8. The method of claim 5, wherein, The first reporter molecule also includes a cleavable group.

9. The method of claim 5, wherein, In the first reporter molecule, the functional group is linked to the cleavable group.

10. The method according to claim 8 or 9, characterized in that, The cleavable groups include photocleavable groups.

11. The method of claim 6, wherein, The second reporter molecule includes at least one of biotin, desulfobiotin, and fluorescent dye.

12. The method of claim 6, wherein, The second reporter molecule also includes a cleavable group.

13. The method of claim 6, wherein, In the second reporter molecule, the functional group is linked to the cleavable group.

14. The method according to claim 12 or 13, characterized in that, The cleavable groups include photocleavable groups.

15. The method of any one of claims 7-9, wherein, After the first click reaction is completed, the protein is digested with a protease to enrich the protein of the first reporter molecule marker, and then lysed.

16. The method of any one of claims 11-13, wherein, After the second click reaction is completed, the protein is digested with a protease to enrich the protein of the second reporter molecule marker, and then lysed.

17. The method of claim 15, wherein, The pyrolysis is photopyrolysis.

18. The method of claim 17, wherein, The photolysis was performed under ultraviolet light conditions.

19. The method of claim 16, wherein, The pyrolysis is photopyrolysis.

20. The method of claim 19, wherein, The photolysis was performed under ultraviolet light conditions.

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