Binding peptide targeting human AARS1 protein C-terminal structural domain and application

Abstract

The invention relates to the technical field of biological medicine, and discloses a binding peptide targeting a C-terminal structural domain of human AARS1 protein and application, the binding peptide comprises an amino acid sequence represented by a general formula (I) and a Val-Ala-Xaa-Xaa-Arg-His core motif, and can specifically bind to the C-terminal structural domain of the human AARS1 protein. The binding peptide and the derivative thereof block an AARS1 mediated protein lactic acid modification signal channel by inhibiting the activity of AARS1 lactoacyltransferase or inducing protein degradation through a ubiquitin-proteasome pathway. The binding peptide provided by the invention avoids an N-terminal catalytic activity center of AARS1, does not influence the protein synthesis function of normal cells while exerting antitumor activity, has relatively high affinity and safety, and can be used for preparing drugs for treating AARS1 mediated diseases such as pancreatic cancer or a diagnostic reagent for detecting AARS1.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a binding peptide targeting the C-terminal domain of human AARS1 protein and application thereof. BACKGROUND

[0002] Post-translational modification of proteins is an important mechanism for regulating cell life activities. Among them, protein lactylation modification, as a modification method connecting cell metabolic state and protein function, converts metabolic product lactic acid into a signal for regulating gene expression, cell proliferation and immune response by covalently introducing lactyl group on lysine residues of proteins. In addition to the classic aminoacylation function, alanyl-tRNA synthetase 1 (AARS1) has been confirmed to have lactyltransferase activity, which can catalyze the lactylation modification of key signaling proteins such as YAP and p53, and plays a role in the occurrence and development of tumor, neurodegenerative disease, fibrosis disease and autoimmune disease.

[0003] Although the lactyltransferase function of AARS1 is a potential target for intervention in the above diseases, the existing technology has limitations in drug development targeting this target. Currently, there is no report of a ligand molecule that can specifically bind to the C-terminal domain of AARS1. Due to the lack of binding molecules targeting this specific domain, it is difficult to achieve specific regulation of the lactylation modification function mediated by AARS1. In addition, the existing inhibitors targeting AARS1 mainly act on its N-terminal catalytic domain, which will inhibit the classic aminoacylation activity of AARS1, interfere with the normal protein synthesis process in cells, and thus cause non-specific cytotoxicity, limiting its application in clinical treatment. Therefore, the development of a binding molecule that can specifically target the C-terminal domain of AARS1 without affecting its classic biological function is of great significance for the treatment of related diseases and drug development. SUMMARY

[0004] The present application provides a binding peptide targeting the C-terminal domain of human AARS1 protein, which adopts the following technical scheme: A binding peptide targeting the C-terminal domain of human AARS1 protein, the binding peptide comprising an amino acid sequence represented by general formula (I): X 1 -Lys-Val-Ala-X 5 -X 6 -Arg-His-X 9 -X 10 -Thr-Ala(I) wherein, X 1 is selected from Ser, Pro, Thr, Val or Arg; X 5 is selected from Gln or Glu; X 6selected from Ala, Ser, Lys, Gly or Arg; X 9 selected from Asp, Asn, Lys, Glu or Arg; X 10 selected from Val or Pro; and the binding peptide comprises a Val-Ala-Xaa-Xaa-Arg-His core motif, capable of specifically binding to the C-terminal domain of human AARS1 protein.

[0005] By adopting the above technical solution, the application realizes the specific binding of the C-terminal domain of human AARS1 protein by using the Val-Ala-Xaa-Xaa-Arg-His (VAxxRH) tripeptide core motif in the binding peptide sequence. The action mechanism and beneficial effects are as follows: Firstly, the binding peptide forms a stable complex with the C-terminal domain of AARS1 protein through the VAxxRH core motif.

[0006] Secondly, the application realizes a separate intervention strategy for the function of AARS1 protein. Unlike the inhibitors in the prior art that target the N-terminal catalytic domain of AARS1, the binding peptide provided by the application does not bind to the N-terminal active center responsible for aminoacylation reaction. Therefore, while blocking the pathological signaling of AARS1 through steric hindrance or inducing degradation, the binding peptide does not inhibit the protein synthesis function of normal cells, reducing the toxicity to normal cells.

[0007] Preferably, the amino acid sequence of the binding peptide is SEQ ID NO: 1: PKVAQSRHDVTA.

[0008] Preferably, the binding peptide serves as a targeting ligand and is coupled with an effector ligand through a linker; the effector ligand is selected from any one of the following: an E3 ubiquitin ligase ligand, an autophagy targeting ligand, a lysosome targeting ligand, a deubiquitinase ligand or a lactate translocase ligand.

[0009] Preferably, the binding peptide is a PROTAC molecule containing an E3 ubiquitin ligase ligand, and the amino acid sequence is as shown in any one of the following: PKVAQSRHDVTAGGSGGALAPYIP; SKVAQKRHDVTAGGSGGALAPYIP; TKVAQARHDVTAGGSGGALAPYIP.

[0010] By adopting the above preferred technical solution, the binding peptide is constructed into a protein degradation targeting chimera (PROTAC) molecule, which induces the degradation of AARS1 protein through the ubiquitin-proteasome pathway. The specific mechanism is as follows: One end of the chimera molecule specifically binds the C-terminal domain of the intracellular AARS1 protein through a binding peptide, and the other end recruits an E3 ubiquitin ligase through an effector ligand; a flexible linker pulls the AARS1 protein and the E3 ubiquitin ligase close, inducing the formation of a ternary complex; in the ternary complex, the E3 ubiquitin ligase catalyzes the transfer of ubiquitin molecules to the surface of the AARS1 protein, causing it to be polyubiquitinated; the ubiquitin-labeled AARS1 protein is then recognized and hydrolyzed by the proteasome in the cell, thereby reducing the intracellular AARS1 protein level.

[0011] Preferably, the binding peptide is in the form of a pharmaceutically acceptable salt; the pharmaceutically acceptable salt is selected from inorganic acid salts or organic acid salts; and further preferably one of trifluoroacetate, acetate, hydrochloride, sulfate or phosphate.

[0012] By using the above preferred technical solution, the binding peptide is prepared into a specific salt type, which can improve the water solubility and stability of the polypeptide molecule, and adapt to the processing and storage requirements of pharmaceutical preparations.

[0013] The application provides an application of a binding peptide targeting the C-terminal domain of human AARS1 protein, which adopts the following technical solution: The application uses the binding peptide targeting the C-terminal domain of human AARS1 protein to prepare a drug for regulating the activity of AARS1 protein or treating AARS1-mediated diseases, or to prepare a diagnostic reagent for detecting the expression level or lactylation modification activity of AARS1 protein.

[0014] By using the above technical solution, based on the specific binding ability of the binding peptide to the C-terminal domain of AARS1, the application in the fields of pharmacy and diagnosis is realized: In the field of pharmacy, the binding peptide and its derivatives can play a role in pathological conditions of abnormal function of AARS1 protein: The binding peptide binds to the C-terminal domain of AARS1 protein; The PROTAC molecule constructed by the binding peptide can mediate the degradation of AARS1 protein and eliminate pathogenic proteins.

[0015] In the field of diagnosis, the specific binding property of the binding peptide to the C-terminal domain of AARS1 protein can be used to identify AARS1 protein from biological samples, providing a tool for disease detection.

[0016] Preferably, the regulation of the activity of AARS1 protein refers to inhibiting the lactoyltransferase activity of AARS1 protein, or inducing the degradation of AARS1 protein through the ubiquitin-proteasome pathway.

[0017] By adopting the above preferred technical scheme, the metabolic signal in the cell is regulated by interfering with the lactyltransferase function of AARS1: The peptide binds to the C-terminal region of AARS1, competitively inhibiting the binding of lactyl-CoA or substrate protein; this inhibition blocks the process of AARS1 catalyzing the transfer of a lactyl group to the substrate protein, reducing the level of lactylation modification of proteins in the cell; as the level of lactylation modification decreases, the transcription of downstream related genes or metabolic pathways is regulated.

[0018] Preferably, the AARS1-mediated disease includes a tumor, a neurodegenerative disease, a fibrotic disease, a metabolic disease, or an autoimmune disease; further preferably, the tumor is pancreatic cancer.

[0019] By adopting the above preferred technical scheme, the drug specifically targets the C-terminal domain of the AARS1 protein to correct the metabolic network imbalance in the disease state. The mechanism of action is that in metabolic diseases such as pancreatic cancer, AARS1 is often highly expressed and mediates abnormal protein lactylation modification, thereby maintaining pathological cell proliferation or function; the binding peptide or its PROTAC derivative of the present application can accurately recognize and occupy the non-canonical functional domain of AARS1, or directly induce degradation of the AARS1 protein. This molecular surgical intervention strategy can cut off the signal transduction pathway centered on lactylation modification, destroy the metabolic microenvironment on which tumor cells or other diseased cells depend for survival, and thus effectively inhibit the progression of the disease without affecting the normal cell protein synthesis housekeeping function. Preferably, the diagnostic reagent comprises a detectable label coupled to the binding peptide, and the detectable label is selected from a fluorescent group, a radioisotope, biotin, or horseradish peroxidase.

[0020] By adopting the above preferred technical scheme, the detectable label is coupled to the binding peptide to become a molecular probe; the probe reflects the expression amount or distribution of AARS1 protein in the sample by detecting the intensity and distribution of the signal.

[0021] The present application provides a binding peptide targeting the C-terminal domain of human AARS1 protein and applications. It has the following advantages: 1、The binding peptide provided by the present application contains a Val-Ala-Xaa-Xaa-Arg-His (VAxxRH) core motif, which can specifically recognize and bind to the C-terminal domain of human AARS1 protein, with a nanomolar-level equilibrium dissociation constant (KD), providing a high-affinity lead molecule for treating AARS1-mediated tumor malignant progression.

[0022] 2、The application adopts the strategy of targeting the C-terminal domain of AARS1, avoiding the N-terminal catalytically active center responsible for aminoacylation reaction. Compared with the existing technology of targeting the N-terminal inhibitor, it can physically block the binding of lactylation modification substrate or induce AARS1 protein degradation, thereby precisely blocking the pathological lactylation modification signal pathway, effectively intervening in the disease signal mediated by AARS1, and not directly inhibiting the protein translation function required for AARS1 to maintain normal cell survival through steric hindrance, so it has higher action specificity and safety window.

[0023] 3、The application constructs a protein degradation targeting chimera (PROTAC) based on the binding peptide, which is coupled with an E3 ubiquitin ligase ligand through a flexible linker, and can induce AARS1 protein to form a ternary complex with E3 ubiquitin ligase. The chimera utilizes the ubiquitin-proteasome pathway to mediate the ubiquitination degradation of AARS1 protein, reduces the expression level of intracellular AARS1 protein, and realizes effective intervention on the non-canonical function of AARS1. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 AARS1 binding peptide No. 1 generated for AlphaFold3 and AARS1-C-terminal domain binding mode diagram; Figure 2 Representative map of polypeptide PROTACs recombinant vector for construction #1; Figure 3 Representative map of polypeptide PROTACs recombinant vector for construction #2; Figure 4 Representative map of polypeptide PROTACs recombinant vector for construction #3; Figure 5 Representative sequencing result diagram of polypeptide PROTACs recombinant vector for successful construction #1; Figure 6 Representative sequencing result diagram of polypeptide PROTACs recombinant vector for successful construction #2; Figure 7 Representative sequencing result diagram of polypeptide PROTACs recombinant vector for successful construction #3; Figure 8 Result diagram and quantitative analysis diagram of Western Blot for detecting AARS1 expression; Figure 9 Sequencing result diagram of P1 polypeptide successfully synthesized in vitro; Figure 10 Result diagram and quantitative analysis diagram of Western Blot for detecting AARS1 expression after treating pancreatic cancer cell line MiaPaca2 with AARS1 Protac polypeptide P1; Figure 11 Figure and quantitative analysis of Western Blot results for detecting AARS1 expression and global global-pantilamination expression after treating mouse hippocampal neuron cell line Ht22 with AARS1 Protac polypeptide P1. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the specification of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0026] Preparation Example 1-2: Preparation Example 1: The present preparation example provides a general preparation method for binding peptides (No. 1, No. 2, No. 3) and targeted degradation chimeras (P1, P2, P3).

[0027] Standard Fmoc solid-phase peptide synthesis process (SPPS) is adopted. According to the sequence of the target polypeptide from C-terminal to N-terminal, it is extended one by one in the solid-phase reactor.

[0028] Resin swelling and loading: According to the sequence length and synthesis difficulty, Wang Resin resin with a substitution degree in the range of 0.4-0.8 mmol / g is selected. An appropriate amount of resin is placed in the reactor, and after swelling in DMF for 30 minutes, it is dried.

[0029] Coupling cycle: The following cycle operation is performed for each amino acid: Deprotection: add 20% (v / v) piperidine in DMF solution, react for 10-20 minutes to remove the Fmoc protecting group, and then wash with DMF for 3 times.

[0030] Condensation: weigh Fmoc-protected amino acid, condensing agent HATU and basic reagent DIEA, dissolve in DMF to activate and then add to the reactor.

[0031] Condition optimization: adjust the reaction parameters according to the characteristics of different sequences to ensure the coupling efficiency: For conventional sequences (such as binding peptide No. 1 / P1), 4 times the amount of amino acid and 3.8 times the amount of HATU are used, and the reaction is carried out at 25°C for 45 minutes; For sequences prone to side reactions (such as binding peptide No. 2 / P2), the reagent concentration is reduced and the temperature is controlled to 20°C, and the reaction time is shortened to 30 minutes; For long chain or sterically hindered sequences (such as binding peptide No. 3 / P3), increase the reagent to 5 equivalents, and increase the temperature to 30°C, and extend the reaction time to 60 minutes.

[0032] Lysis and purification: After synthesis, the resin is shrunk dried with methanol. According to the type of side chain protection group, prepare a lysis solution with a volume ratio of TFA:TIS:H2O of 90:5:5 to 95:2.5:2.5, and lysis at room temperature for 1.5-3 hours. Collect the filtrate, precipitate with ice anhydrous ether, and centrifuge to dryness to obtain the crude peptide. Purify the crude product by preparative HPLC (C18 column, gradient elution with acetonitrile / water system), and freeze-dry to obtain the target product as a white powder with a purity of ≥98%.

[0033] Preparation Example 2: This preparation example provides a method for constructing recombinant expression plasmids (#1, #2, #3) encoding the above-mentioned polypeptide PROTACs.

[0034] Sequence design and synthesis: According to the sequences of the binding peptides (No. 1, No. 2, No. 3) and the effector ligand, design the coding DNA sequence containing a flexible linker and a start codon. The specific sequences are as follows: Construct #1 (corresponding to P1): 5'-ATGCCTAAGGTGGCTCAGTCTAGGCACGATGTGACAGCTGGCGGCTCTGGCGGCGCTTTGGCTCCATACATTCCA-3' Construct #2 (corresponding to P2): 5'-ATGAGCAAGGTGGCACAGAAGCGCCACGACGTGACCGCAGGTGGCTCTGGCGGAGCACTGGCTCCATACATTCCA-3' Construct #3 (corresponding to P3): 5'-ATGACCAAGGTGGCACAGGCTAGACACGACGTCACCGCTGGAGGCAGCGGCGGAGCACTGGCTCCATACATTCCA-3' Vector construction: Select pVax-1 as the basic expression vector. Linearize the vector multiple cloning site with restriction enzymes, and insert the above-mentioned double-stranded DNA fragments into the vector by homologous recombination. Transform the E. coli competent cells, select single clones for amplification, and extract the plasmid.

[0035] Sequencing verification: The extracted recombinant plasmid was verified by Sanger sequencing method. The sequencing results confirmed that the inserted fragment sequence was correct, the reading frame was not shifted or mutated, and the recombinant plasmids #1, #2 and #3 were successfully obtained.

[0036] Example 1-3: Example 1: The present example provides a binding peptide No. 1 targeting the C-terminal domain of human AARS1 protein and its derived targeted degradation chimera P1, as well as a recombinant plasmid encoding the chimera, comprising the following steps: Chemical synthesis of polypeptide: Referring to the attached Figure 1 , according to the parameters of Preparation Example 1, 0.6 mmol / g Wang Resin was used as the carrier, 25°C condensation, TFA:TIS:H2O=95:2.5:2.5 cleavage. After HPLC purification and freeze-drying, the binding peptide No. 1 (sequence: PKVAQSRHDVTA) and the targeted degradation chimera P1 (sequence: PKVAQSRHDVTAGGSGGALAPYIP) were obtained. Mass spectrometry detection showed that the molecular weight was consistent with the theoretical value (see Figure 9 ).

[0037] Construction of recombinant plasmid: According to the amino acid sequence of the chimera P1, the coding DNA sequence was designed, and the sequence was as follows: 5'-ATGCCTAAGGTGGCTCAGTCTAGGCACGATGTGACAGCTGGCGGCTCTGGCGGCGCTTTGGCTCCATACATTCCA-3' The pVax-1 vector was selected, and the above DNA sequence was inserted into the vector multiple cloning site by molecular cloning technology. Sequencing verification (see Figure 5 results) showed that the sequence was correctly connected, and the recombinant plasmid #1 was obtained, and the spectrum is shown in Figure 2 .

[0038] Example 2: The present example provides a binding peptide No. 2 targeting the C-terminal domain of human AARS1 protein and its derived targeted degradation chimera P2, as well as a recombinant plasmid encoding the chimera, comprising the following steps: Chemical synthesis of polypeptide: Preparation was carried out according to the parameters described in Preparation Example 1.

[0039] Construction of recombinant plasmid: According to the amino acid sequence of the chimera P2, the coding DNA sequence was designed, and the sequence was as follows: 5'-ATGAGCAAGGTGGCACAGAAGCGCCACGACGTGACCGCAGGTGGCTCTGGCGGAGCACTGGCTCCATACATTCCA-3' The above DNA sequence was inserted into the pVax-1 vector. After sequencing verification (see the results of Figure 6 ), the recombinant plasmid #2 was obtained, the map of which is shown in Figure 3 .

[0040] Example 3: This example provides a binding peptide No. 3 targeting the C-terminal domain of human AARS1 protein and a target degradation chimeric P3 derived therefrom, as well as a recombinant plasmid encoding the chimeric, comprising the following steps: Chemical synthesis of polypeptide: preparation was carried out according to the parameters described in Preparation Example 1.

[0041] Construction of recombinant plasmid: According to the amino acid sequence of the chimeric P3, the coding DNA sequence was designed, and the sequence is as follows: 5'-ATGACCAAGGTGGCACAGGCTAGACACGACGTCACCGCTGGAGGCAGCGGCGGAGCACTGGCTCCATACATTCCA-3' The above DNA sequence was inserted into the pVax-1 vector. After sequencing verification (see the results of Figure 7 ), the recombinant plasmid #3 was obtained, the map of which is shown in Figure 4 .

[0042] Test Example 1-2: Test Example 1: Experimental steps: Human pancreatic cancer cell line MiaPaca2 was selected as the test object. Cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 3×10 5 cells / well, and incubated in a 37℃, 5% CO2 incubator with DMEM medium containing 10% fetal bovine serum overnight to adhere.

[0043] Subsequently, the following two treatments were carried out: Treatment 1: When the cell confluence reached about 50%, transfection was performed. One hour before transfection, the old medium was discarded and replaced with fresh complete medium without antibiotics (antibiotics would affect the transfection efficiency and increase cell toxicity). According to the instructions of the commercial transfection reagent used (such as Lipofectamine 2000, PEI, etc.), the plasmid-transfection reagent complex was prepared. The chimeric plasmid #1, #2, #3 prepared in Examples 1, 2, 3 and the control plasmid were mixed with the transfection reagent according to the recommended ratio, and incubated at room temperature (usually 15-20 minutes) to form a complex. The prepared transfection complex was evenly added to the corresponding gradient of cell culture medium, and gently shaken. The cells were placed back in the incubator for continuous culture. Fresh complete medium was replaced 8 hours after transfection.

[0044] After 48 hours of plasmid transfection, the medium was aspirated and the cells were washed twice with pre-cooled PBS buffer. RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes. Centrifuged at 4°C, 12000 rpm for 15 minutes, and the supernatant was collected. The protein concentration was determined by BCA method and quantitatively adjusted.

[0045] Treatment 2: When the cell confluence reached about 70%, fresh medium was replaced. Chimeric P1 prepared in Example 1 was added, and concentration gradients (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) were set, and a blank control group with the same amount of DMSO solvent was also set.

[0046] After 24 hours of drug treatment, the medium was aspirated and the cells were washed twice with pre-cooled PBS buffer. RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes. Centrifuged at 4°C, 12000 rpm for 15 minutes, and the supernatant was collected. The protein concentration was determined by BCA method and quantitatively adjusted.

[0047] After completing Treatment 1 or Treatment 2, an equal amount of protein sample (20 μg) was subjected to 10% SDS-PAGE gel electrophoresis separation, and then wet transferred to a PVDF membrane. Blocked with 5% skim milk at room temperature for 1 hour, and incubated with rabbit anti-human AARS1 primary antibody (1:1000 dilution) and mouse anti-human β-actin primary antibody (1:5000 dilution, internal control) respectively, 4°C overnight. After TBST washing, incubate HRP-labeled secondary antibody at room temperature for 1 hour. Add ECL chemiluminescence solution and expose to imaging system for imaging.

[0048] ImageJ software was used to perform grayscale analysis on protein bands. The AARS1 band was normalized based on the grayscale of the β-actin band, and the relative degradation rate of the protein at different concentrations was calculated. The half-maximum degradation concentration (DC50) and the maximum degradation rate (Dmax) were calculated using GraphPad Prism software [Dmax=(1-grayscale value of treatment group / grayscale value of control group)x100%].

[0049] Experimental data: Data on AARS1 degradation induced by plasmid-synthesized chimeric molecules in MiaPaca2 cells are shown in Table 1.

[0050] Table 2 shows the data on AARS1 degradation induced by chemically synthesized polypeptide chimeric molecules in MiaPaca2 cells.

[0051] Table 1. Degradation activity parameters of plasmid-synthesized chimeric molecules on intracellular AARS1 protein

[0052] Table 2. Degradation activity parameters of the polypeptide chimeric molecule P1 on intracellular AARS1 protein

[0053] Note: A higher Dmax value indicates a more thorough maximum degradation.

[0054] Experimental conclusion: Plasmid transfection results: such as Figure 8 As shown, compared with the control group, the expression of AARS1 protein was significantly downregulated in cells transfected with the recombinant plasmids #1, #2, and #3 of this invention (p<0.0001). This result confirms that the PROTAC polypeptide produced intracellularly through gene expression also has the function of inducing target protein degradation.

[0055] Results of peptide treatment: as shown in Table 2 and Figure 10 As shown, after treatment with chimeric P1, the expression level of AARS1 protein in MiaPaca2 cells decreased significantly with increasing concentration (p<0.0001), confirming the biological activity of the chemically synthesized peptide.

[0056] Test Example 2: Experimental steps: This experiment aims to further verify, from a functional perspective, the mechanism by which the binding peptide of this invention blocks the downstream lactation modification signaling pathway by mediating AARS1 degradation, and to confirm the decisive role of the VAxxRH core motif in this process.

[0057] The experimental cells were selected from Ht-22 cell line. The cells were inoculated in 6-well plates and cultured to 70% confluence. The chimeric body P1 prepared in Example 1 was added at different concentrations (0 μM, 12.5 μM, 25 μM, 50 μM, 100 μM), respectively.

[0058] After 24 hours of drug incubation, the cells were collected and total protein was extracted. After quantification by BCA method, equal amount of protein (30 μg) was subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. After blocking, the specific antibody for pan-kla modification (Pan-Kla Antibody, 1:1000 dilution) and β-actin internal reference antibody were incubated, respectively. After washing and incubation with secondary antibody, chemiluminescence color development was performed.

[0059] The cumulative gray value of the whole protein lactylation band in the lane was determined by optical density analysis software, and the β-actin was used as the internal reference for correction, and the relative level of protein lactylation modification of each group relative to the blank control group was calculated.

[0060] Experimental data: The relative level of whole protein lactylation modification of each group of cells treated with different chimeric body molecules is shown in Table 3.

[0061] Table 3. Determination of whole protein lactylation modification level of Ht-22 cells treated with P1 chimeric body molecules

[0062] Note: The relative gray value of whole protein lactylation reflects the abundance of whole protein lactylation modification in cells; the lower the value, the more obvious the inhibition effect.

[0063] Experimental conclusion: As shown in Table 3 and Figure 11 After treatment with Example 1 (P1), the whole protein lactylation modification level of Ht-22 cells was significantly reduced. This result is consistent with the function of AARS1 as a lactyltransferase, indicating that the chimeric body blocks the lactyl group transfer pathway by degrading AARS1 protein, thereby inhibiting the global lactylation level.

[0064] Based on the experimental results of human pancreatic cancer cells MiaPaca2 and murine neuronal cells Ht-22, the following conclusions were drawn: Mechanism universality: whether in human tumor cells or murine neuronal cells, the chimeric body P1 can effectively block the lactyl group transfer pathway by degrading AARS1 protein, thereby inhibiting the global lactylation level.

[0065] Cross-species and multi-tissue applicability: The binding peptide described in the present application shows significant activity in both human and mouse cells of different species, as well as in cells of different tissue sources of pancreas and nerve. This strongly confirms the high conservation of the C-terminal domain of AARS1 (especially the VAxxRH core motif) among mammals, indicating that the drug molecule has broad application potential, not only suitable for tumor treatment, but also suitable for neurodegenerative diseases and other pathological processes mediated by AARS1.

[0066] In summary, the test demonstrates the structure-activity relationship of the technical scheme: targeting the C-terminal of AARS1 with the VAxxRH core motif, down-regulating the protein lactylation modification level in cells by degrading AARS1, and the mechanism is effective in different species and tissues.

[0067] SEQUENCE NUMBER:

Claims

1. A binding peptide targeting the C-terminal domain of human AARS1 protein, characterized in that, Contains an amino acid sequence represented by general formula (I): X 1 -Lys-Val-Ala-X 5 -X 6 -Arg-His-X 9 -X 10 -Thr-Ala(I) Among them, X 1 Selected from Ser, Pro, Thr, Val, or Arg; X 5 Selected from Gln or Glu;X 6 Selected from Ala, Ser, Lys, Gly, or Arg; X 9 Selected from Asp, Asn, Lys, Glu, or Arg; X 10 Choose from Val or Pro; Furthermore, the binding peptide contains the Val-Ala-Xaa-Xaa-Arg-His core motif, which can specifically bind to the C-terminal domain of the human AARS1 protein.

2. The binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 1, characterized in that, The amino acid sequence of the binding peptide is SEQ ID NO: 1: PKVAQSRHDVTA.

3. The binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 1, characterized in that, The binding peptide, as a targeting ligand, is coupled to the effector ligand via a linker. The effector ligand is selected from any one of the following: E3 ubiquitin ligands, autophagy-targeting ligands, lysosome-targeting ligands, and deubiquitinated enzyme ligands constitute a protein hydrolysis-targeting chimera, an autophagy-targeting chimera, a lysosome-targeting chimera, and a deubiquitinated targeting chimera.

4. The binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 1, characterized in that, The binding peptide is a PROTAC molecule containing an E3 ubiquitin ligase ligand, and its amino acid sequence is shown in any of the following: PKVAQSRHDVTAGGSGGALAPYIP; SKVAQKRHDVTAGGSGGALAPYIP; TKVAQARHDVTAGGSGGALAPYIP.

5. The binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 1, characterized in that, The binding peptide is one of trifluoroacetate, acetate, hydrochloride, sulfate, or phosphate.

6. The application of a binding peptide targeting the C-terminal domain of human AARS1 protein, characterized in that, The use of the binding peptide targeting the C-terminal domain of human AARS1 protein as described in any one of claims 1 to 5 in the preparation of a medicament for regulating AARS1 protein activity or for treating AARS1-mediated diseases, or in the preparation of a diagnostic reagent for detecting AARS1 protein expression levels or lactation modification activity.

7. The application of the binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 6, characterized in that, The regulation of AARS1 protein activity refers to: Inhibit the lactyltransferase activity of AARS1 protein, or induce AARS1 protein degradation via the ubiquitin-proteasome pathway.

8. The application of the binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 6, characterized in that, The diseases mediated by AARS1 include tumors, neurodegenerative diseases, fibrotic diseases, metabolic diseases, or autoimmune diseases.

9. The application of the binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 8, characterized in that, The tumor is pancreatic cancer; The drug is the Protac molecule described in claim 4, which inhibits tumor cell proliferation by specifically binding to the C-terminal domain of the AARS1 protein and blocking or downregulating the level of protein lactation modification mediated by the AARS1 protein.

10. The application of the binding peptide targeting the C-terminal domain of human AARS1 protein according to claim 6, characterized in that, The diagnostic reagent contains a detectable marker conjugated to the binding peptide, the detectable marker being selected from fluorescent groups, radioisotopes, biotin, or horseradish peroxidase.

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