Cyclic peptide inhibitors of aminoglycoside phosphotransferase and uses thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an aminoglycoside phosphotransferase cyclopeptide inhibitor and application thereof. APH is a key enzyme capable of causing inactivation of aminoglycoside antibiotics. Based on a phage display technology, a series of polypeptides containing two cysteine residues are successfully screened out, and candidate cyclopeptide molecules are generated after the polypeptides are modified by a chemical cross-linking agent. Fluorescence polarization analysis and enzyme activity determination results show that the binding affinity of the cyclopeptide molecules and the target protein APH reaches a nanomole level or a low micromole level, and the catalytic activity of the cyclopeptide molecules can be remarkably inhibited. The development of the cyclopeptide inhibitor provides a new research basis for further optimization and clinical application of APH targeted drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an aminoglycoside phosphotransferase cyclic peptide inhibitor and a use thereof. Background Art

[0002] Antibiotic resistance is one of the major challenges facing global public health. Resistance to aminoglycoside antibiotics mainly stems from the catalytic action of aminoglycoside-modifying enzymes (AMEs), which chemically modify antibiotics to make them lose their antibacterial activity. Among them, aminoglycoside phosphotransferases (APHs), as a typical AME, can transfer the phosphate group of ATP to the hydroxyl group on the antibiotic molecule, causing it to lose its antibacterial activity, such as APH(3')-I.

[0003] In order to cope with the increasingly serious problem of bacterial resistance, in addition to reducing the use of antibiotics, developing new antibiotics and strengthening the monitoring of drug-resistant bacteria, designing new inhibitors against AME is an important research strategy. Ideal AME inhibitors should be able to bind tightly to the target enzyme, thereby protecting aminoglycoside antibiotics from binding to the ribosome A site, inhibiting bacterial protein translation and preventing its proliferation. However, although the research on AME inhibitors has been carried out for more than 40 years, there are currently no reports on cyclic peptide AME inhibitors, nor have any AME targeted inhibitors been used in clinical practice.

[0004] Aminoglycoside phosphotransferase (APH) is a well-studied AME. Due to its structural similarity with eukaryotic protein kinases (ePKs), some ePK inhibitors (such as anthrapyrazolone, 4-Anilinoquinazoline and pyrazolopyrimidine compounds) also have inhibitory effects on APH. Related studies have resolved the crystal structure of the complex of these inhibitors with APH, and found that pyrazolopyrimidine derivatives can significantly inhibit the activity of APH(3')-Ia and restore the antibacterial effect of antibiotics on drug-resistant Escherichia coli. However, in order to achieve clinical application, these inhibitors still need to be further optimized to avoid affecting the normal physiological functions of the host while retaining the inhibitory activity.

[0005] Peptide drugs have attracted much attention in drug development due to their easy synthesis, significant pharmacological activity, and efficient interaction with protein targets. Compared with linear peptides, cyclic peptides have higher affinity, specificity, stability, and cell membrane permeability. The diverse cyclization modes of cyclic peptides (such as disulfide bonds, amide bonds, and other covalent bonds) give them unique advantages in drug development. Currently, the FDA has approved more than 40 cyclic peptide drugs for marketing, including cyclosporin, desmopressin, and anidulafungin.

[0006] Although many advances have been made in the field of cyclic peptide drugs, the development of cyclic peptide inhibitors for aminoglycoside phosphotransferase (APH) is still a blank. The development of cyclic peptide molecules with high affinity for APH and efficient inhibition of its activity not only fills the research gap in this field, but also provides important molecular tools and new ideas for the treatment of drug-resistant bacteria. Summary of the invention

[0007] Developing drug-resistant enzyme inhibitors is a method to solve the problem of bacterial resistance. The present invention provides aminoglycoside phosphotransferase cyclic peptide inhibitors and uses thereof. The present invention uses aminoglycoside phosphotransferase as a target protein to screen and obtain a series of cyclic peptide inhibitors, laying a molecular foundation for the subsequent development and application of aminoglycoside phosphotransferase cyclic peptide inhibitors.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The aminoglycoside phosphotransferase cyclic peptide inhibitor is selected from one of the polypeptides having the following amino acid sequence:

[0010] (1) Ala-Cys-Ser-Trp-Pro-Leu-Cys-Glu-Leu-Leu (sequence shown in SEQ ID NO. 1);

[0011] (2) Cys-Gly-Trp-Leu-Leu-Cys-Pro-Phe-Val (sequence shown in SEQ ID NO. 2);

[0012] (3) Ile-Cys-Ala-Trp-Pro-Leu-Cys-Asn-Leu-Leu (sequence shown in SEQ ID NO. 3);

[0013] (4) Cyclic peptides containing the conserved sequence Cys-X-Trp-X-Leu-Cys, where X is any amino acid;

[0014] (5) A cyclic peptide containing the conserved sequence Cys-Pro-X-Tyr-Cys, wherein X is any amino acid.

[0015] In one embodiment of the present invention, the aminoglycoside phosphotransferase cyclic peptide inhibitor is selected from one of the molecules A-L2, A-L3, A-L4, A-L7, C-L2, C-L3, C-L4, I-L2, I-L3, I-L4, I-L7 or Y-L5:

[0016]

[0017]

[0018]

[0019] The aminoglycoside phosphotransferase cyclic peptide inhibitors provided by the present invention all contain two cysteines. The side chain thiol of the cysteine ​​in the polypeptide undergoes an affinity substitution reaction with the active halogen Br on the chemical cross-linking agent, thereby modifying the cyclic peptide. The amino acid sequences of the cyclic peptides A-L2, A-L3, A-L4, A-L7, C-L2, C-L3, C-L4, I-L2, I-L3, I-L4, I-L7 and Y-L5 of the present invention and their binding affinity to APH are specifically shown in Table 1.

[0020] Table 1 APH candidate cyclic peptide inhibitor sequences and binding affinities

[0021]

[0022] The present invention further provides a method for obtaining a cyclic peptide molecule specifically targeting APH, which can be obtained by phage display technology. First, the target protein APH is purified in vitro, and then 10 11 The candidate cyclic peptides of the target protein were screened from the phage display library of the order of magnitude. The screening process included the steps of "preparation and blocking of the phage cyclic peptide library-binding of the target protein to the magnetic beads and blocking-phage display screening". Multiple rounds of screening were performed by increasing the screening pressure in different screening rounds, and the phage titer after each round of screening was measured by infection counting. The specific binding phages enriched in each round of screening were sequenced using the Illumina second-generation sequencing platform, and the second-generation sequencing data were processed by Matlab scripts to obtain the sequence and abundance of the target APH-specific binding peptide.

[0023] The present invention further provides a method for modifying and purifying the cyclic peptide molecule that specifically targets APH. In one embodiment of the invention, the method for modifying and purifying the cyclic peptide molecule that specifically targets APH is as follows: the modification reaction system is 100mM NH4HCO3 (pH 8.0), 50% ACN, 0.5mM polypeptide and 1mM chemical cross-linking agent, and the reaction is carried out at 30°C for 1h. The pure cyclic peptide molecule is obtained by HPLC purification and preparation (the mobile phase is pure water (containing 0.1% TFA) and CAN (containing 0.1% TFA)), and the molecular weight is verified by mass spectrometry and then lyophilized.

[0024] The present invention uses fluorescence polarization technology to determine the binding affinity between the cyclic peptide molecule and the target protein APH. According to the measurement, the affinity between the cyclic peptide of the present invention and APH is low micromolar, which lays a foundation for the development of cyclic peptide inhibitor molecules targeting APH.

[0025] The present invention further provides an isolated nucleic acid encoding the aminoglycoside phosphotransferase cyclic peptide inhibitor.

[0026] The present invention further provides a vector, which comprises the nucleic acid.

[0027] The present invention further provides a pharmaceutical composition, which comprises the aminoglycoside phosphotransferase cyclic peptide inhibitor (cyclic peptide molecule specifically targeting APH) and one or more pharmaceutically acceptable excipients and / or carriers.

[0028] The present invention further provides the use of the aminoglycoside phosphotransferase cyclic peptide inhibitor (cyclic peptide molecule specifically targeting APH) in preparing a therapeutic drug for aminoglycoside antibiotic-resistant bacteria.

[0029] More specifically, the present invention provides the use of the aminoglycoside phosphotransferase cyclic peptide inhibitor (cyclic peptide molecule specifically targeting APH) in the preparation of a therapeutic drug for treating drug-resistant bacteria associated with APH expression.

[0030] The present invention further provides the use of the aminoglycoside phosphotransferase cyclic peptide inhibitor (cyclic peptide molecule specifically targeting APH) in the preparation of a therapeutic drug for treating diseases related to the expression of protein kinases similar to APH sequences. Compared with the prior art, the present invention has the following advantages and effects:

[0031] The present invention provides cyclic peptide molecules that specifically target APH and uses thereof. Aminoglycoside phosphotransferase APH is an aminoglycoside antibiotic-resistant enzyme, and the development of resistant enzyme inhibitors is an important way to treat drug-resistant bacteria. The present invention uses phage display technology, which is widely used in the development of peptide or antibody drugs, to carry out multiple rounds of screening for APH protein, and performs second-generation sequencing on the enriched sequences to obtain candidate cyclic peptide molecule sequences that specifically bind to APH. The cyclic peptide molecules all contain two cysteines, which are modified into rings by specific chemical cross-linking agents. The affinity of the cyclic peptide molecules to the target protein APH is determined by fluorescence polarization technology to be at the low micromolar level, and experimental measurements have found that the cyclic peptides exhibit an inhibitory effect on the enzymatic activity of APH. The cyclic peptides in this study lay the foundation for the development of inhibitors targeting APH.

[0032] (1) Compared with traditional antibodies or small molecule inhibitors, the cyclic peptide molecules of the present invention have the advantages of high affinity, strong targeting, small size, easy synthesis and modification, low cost, simple operation, and flexible administration route.

[0033] (2) The cyclic peptide molecule of the present invention is reported for the first time at home and abroad and is innovative.

[0034] (3) The 12 cyclic peptides specifically targeting APH provided by the present invention can be used as therapeutic drugs for aminoglycoside antibiotic-resistant bacteria and have potential clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments so that those skilled in the art can better understand the present invention and implement it.

[0036] Figure 1 :The enrichment degree of candidate cyclic peptides specifically targeting APH during the screening process was determined based on the phage titer.

[0037] Figure 2 : The peptide sequences and abundances specifically targeting APH were obtained through three rounds of phage display screening under four cross-linking agent conditions.

[0038] Figure 3 : Experimental results of characterizing the binding affinity of 12 cyclic peptide molecules with the target protein APH using fluorescence polarization technology.

[0039] Figure 4 : The HPLC method was used to determine the change in the content of the product ADP before and after the reaction, and the experimental results of the inhibition of the target protein APH enzyme activity by the candidate cyclic peptide molecule AR-L3 at 0.31-5.00 μM were determined. DETAILED DESCRIPTION

[0040] The main materials, reagents and formulations in the following examples of the present invention are shown in Table 2.

[0041] Table 2 Main experimental materials and reagents in the examples

[0042]

[0043]

[0044] The main reagent formulas of the following embodiments of the present invention are as follows:

[0045] 1. 10mg / mL tetracycline (10mL): Dissolve 100mg tetracycline powder in 50% ethanol to 10mL. Filter with a 0.22μm filter membrane to sterilize, then dispense into 1.5mL centrifuge tubes and store at -20℃ for a long time.

[0046] 2. 2YT medium (1L): 16g / L peptone, 5g / L NaCl, 10g / L yeast extract. Add 15g / L agar to 2YT solid medium. Autoclave at 121℃ for 20min.

[0047] 3. Buffer N (1L): 200g polyethylene glycol 6000, 146.1g NaCl, dilute to 1L with distilled water, store at 4℃ for a long time.

[0048] 4. Buffer R (1L): 1.58g NH4HCO3, 1.86g EDTA, pH=8.0, make up to 1L with distilled water.

[0049] 5. 20mM TCEP (5mL): 28.7mg TCEP, dilute to 5mL with distilled water, and store at -20℃ for a long time.

[0050] 6. Preparation method of 10mM chemical crosslinker solution: 1,4-di(bromomethyl)benzene (Linker 1 for short), 1,3-di(bromomethyl)benzene (Linker 2), 2,6-di(bromomethyl)pyridine (Linker 3), α,α'-dibromo-o-xylene (Linker 4), trans-1,4-dibromo-2-butene (Linker 5) and 1,3-dibromoacetone (Linker 7) crosslinker solutions are prepared with acetonitrile solution, and 2,6-bis(bromomethyl)naphthalene (Linker 6) crosslinker solution is prepared with DMSO.

[0051] 7. Wash Buffer 1 (1 L): 1.21 g Tris-Cl, 8.77 g NaCl, 2.03 g MgCl2, 0.11 g CaCl2, adjust pH to 7.4, and make up to 1 L with distilled water.

[0052] 8. Wash Buffer 2 (10mL): 10mL Wash Buffer 1, 100mg BSA and 10μL Tween20.

[0053] 9. Wash Buffer 4 (30mL): 30mL Wash Buffer 1, 30μL Tween 20.

[0054] The primer sequences used in the following examples of the present invention are shown in Table 3:

[0055] Table 3 Primer list for preparing NGS library in the embodiment of the present invention

[0056]

[0057]

[0058] Example 1

[0059] Screening and identification of APH-targeting random cyclic peptide libraries using phage display:

[0060] According to the screening steps of "preparation of phage cyclic peptide library and blocking - binding of target protein to magnetic beads and blocking - phage display screening", a total of 3 rounds of screening were performed. By reducing the amount of protein used to gradually increase the screening stringency, Streptavidin magnetic beads and Neutravidin magnetic beads were used alternately in each round of screening to reduce nonspecific binding during the screening process, as shown in Table 4. Among them, Neutravidin magnetic beads were prepared according to the instructions (2 mg Neutravidin protein and 3350 μL 30 mg / mL Dynabeads TM M-280 Tosylactivated incubation).

[0061] Table 4 The immobilization amount of target protein and the type of magnetic beads used in each round of phage screening

[0062]

[0063] 1. Preparation and blocking of phage cyclic peptide library

[0064] 1) Take 1 mL of Escherichia coli TG1 glycerol frozen bacterial solution containing the phage library described in Table 2 (a total of 11 libraries) and inoculate it into 250 mL of 2YT liquid culture medium (containing 10 μg / mL tetracycline), 30°C, 200 rpm, and culture for 9-15 h;

[0065] 2) Collect all culture medium and centrifuge at 4°C, 12000×g, 20min;

[0066] 3) Collect the supernatant, add 25% volume of pre-cooled buffer N, and place on ice for 30 minutes;

[0067] 4) Centrifugation at 4°C, 6500 rpm, 45 min;

[0068] 5) Discard the supernatant, resuspend the phage in 40 mL buffer R, and centrifuge (5000 rpm, 30 min) to remove bacterial cells;

[0069] 6) Add 2 mL of 20 mM TCEP to 40 mL of phage solution and leave at room temperature for 30 min;

[0070] 7) Add 25% volume of pre-cooled buffer N, place on ice for 10 min, and centrifuge at 4°C, 4500 rpm, 15 min;

[0071] 8) Discard the supernatant, resuspend the phage in 36 mL of buffer R, and then divide into eight equal portions of phage solution;

[0072] 9) Add 0.5 mL of the corresponding chemical crosslinker (100 mM Linker) to each 4.5 mL of phage solution and incubate in a 30°C water bath for 1 h;

[0073] 10) Add 25% volume of pre-cooled buffer N, place on ice for 10 min, and centrifuge at 4°C, 4500 rpm, 15 min;

[0074] 11) Discard the supernatant, resuspend the phage in 5 mL of Wash buffer 2, and place on a rotating mixer (10 rpm, 30 min) to block the phage.

[0075] 2. Binding and blocking of target protein and magnetic beads

[0076] 1) Take 160 μL of magnetic bead solution stored at 4°C, wash twice with 1 mL of Wash buffer 1 solution, and resuspend with Wash buffer 1 solution;

[0077] 2) Add 4 μg APH(3')-Biotin, mix well, and place on a rotating mixer (10 rpm, 10 min);

[0078] 3) Discard the supernatant, wash twice with 1 mL Wash buffer 1, and then add 400 μL Wash buffer 2 to resuspend the magnetic beads;

[0079] 4) Place the mixture on a rotating mixer (10 rpm, 30 min) to block the target protein.

[0080] 3. Phage display screening

[0081] 1) 5 mL of phage solution obtained in step 1 and 50 μL of target protein (with magnetic beads) sample obtained in step 2 were mixed respectively, and placed in a rotating mixer (10 rpm, 1 h) for binding reaction;

[0082] 2) Remove the supernatant, wash 8 times with Wash buffer 4, and then wash 2 times with Wash buffer 1 to remove unbound phages;

[0083] 3) Collect the magnetic beads and resuspend them in 400 μL of the corresponding buffer solution according to the experimental design;

[0084] 4) Transfer 5 mL of E. coli TG1 solution (OD 600 =0.5-0.8), 37℃ infection for 30min;

[0085] 5) 4500rpm, 10min, collect the cells, resuspend with 500μL 2YT medium, take 10μL of the bacterial solution and add it to 90μL 2YT medium, then dilute 7 times by 10 times, take 5μL of each gradient dilution and spot on 2YT solid medium (containing 10μg / mL tetracycline) plate, incubate at 37℃ overnight. Spread the remaining bacterial solution on 2YT medium plate (containing 10μg / mL tetracycline).

[0086] 4. Multiple rounds of phage display screening

[0087] After the first round of phage display screening, 2-3 more rounds of screening are required to increase the abundance of candidate cyclic peptides that bind to the target protein. On the one hand, the main experimental steps such as cyclic peptide library preparation and blocking, target protein fixation and blocking, and screening are repeated. On the other hand, the screening conditions are gradually tightened, such as reducing the amount of target protein, and alternating between streptavidin magnetic beads and neutravidin magnetic beads in multiple rounds of screening (to reduce nonspecific binding during the screening process).

[0088] 5. Next-generation sequencing and analysis of enriched samples

[0089] The recombinant phages obtained from multiple rounds of screening were subjected to two rounds of PCR. The purpose of the first round of PCR was to add specific barcode sequences on both sides of each sample and introduce an index complementary sequence. The purpose of the second round of PCR was to obtain consistent index sequences for the samples. The PCR products were detected by electrophoresis using 2.5% agarose gel. After completing two rounds of reactions, the PCR samples were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing using the Illumina Novaseq 2×150bp sequencing platform.

[0090] 1) First round of PCR:

[0091] The primer sequences used are shown in Table 5, and the experimental samples and corresponding primer pairs are shown in Table 5.

[0092] Table 5 Comparison table of samples enriched in each round of screening and primers in the first round

[0093]

[0094] The PCR reaction system was Template (2.0 μL), 2×PCR NG Master Mix (25 μL), Forward / Reverse Primer (5.0 μL×1 μM), and ddH2O (13.0 μL).

[0095] The PCR program was 95°C × 5 min, (95°C × 30 s, 55°C × 30 s, 72°C × 30 s) × 25 cycles, 72°C × 10 min. The PCR products were subjected to DNA electrophoresis in 2.5% agarose gel to confirm the band size.

[0096] 2) Second round of PCR:

[0097] The products of the first step PCR were mixed in proportion according to the brightness of the bands and used as templates for the second step PCR. 2×NGMaster Mix was used for PCR reaction.

[0098] The PCR reaction system was Template (2.0 μL), 2×NG Master Mix (25.0 μL), Primer S505 / Primer N704 (1.0 μL×5 μM), and ddH2O (21.0 μL).

[0099] The PCR program was 95°C×5min, (95°C×30s, 55°C×60s, 72°C×30s)×25 cycles, 72°C×10min.

[0100] The remaining products of the second round of PCR were mixed and purified using an agarose gel purification kit. The final concentration was determined using a Qubit instrument for next-generation sequencing.

[0101] 3) The second-generation sequencing was completed by GENEWIZ Biotechnology Co., Ltd. on the Illumina Novaseq 2×150bp sequencing platform. The sequencing process mainly includes the construction of the sequencing library, bridge PCR amplification and denaturation, and sequencing. After the sequencing was completed, about 10 million sequencing result data with a length of about 240bp were obtained.

[0102] 4) Data analysis

[0103] The script for analyzing NGS data is based on the original Matlab script in the literature (DOI: 10.1021 / acschembio.4c00099), and the data analysis has been optimized. The content of NGS sequencing data analysis mainly includes data sorting and sequence translation according to specific primer sequences. On this basis, the peptide sequences are clustered and analyzed to obtain more accurate and effective candidate peptide sequences. The enrichment results and abundance of peptide sequences enriched under Linker 2, Linker 3, Linker 4 and Linker 5 are shown in the figure. Figure 2 shown.

[0104] 6. Modification and purification of cyclic peptide molecules

[0105] Order two linear peptide sequences containing two cysteines as described in Table 1 from a peptide synthesis company. Each 0.5 mg peptide was completely dissolved with 500 μL of 50% ACN / 50% Water (containing 0.1% TFA). The modification reaction system was 100 mM NH4HCO3 (pH 8.0), 50% ACN, 0.5 mM peptide and 1 mM chemical cross-linking agent, and the reaction was carried out at 30°C for 1 h. The target cyclic peptide molecules were separated, purified and prepared by Agilent HPLC (chromatographic column: Poroshell 120EC-C18 (4.6×150 mm, 4 μm)), and the molecular weight of the purified product was verified by Shimadzu UPLC-MS (chromatographic column: Shim-pack GISTC18 (2.1×50 mm, 2 μm)), and the correct cyclic peptide was vacuum frozen and lyophilized.

[0106] 7. Determination of the binding ability of candidate cyclic peptides to target protein APH

[0107] In this example, fluorescence polarization was used to characterize the binding ability of the candidate cyclic peptide to the target protein APH.

[0108] The cyclic peptide to be tested was diluted to 100 nM with a buffer solution (containing 0.01% Tween 20). APH was diluted to a certain concentration with the same buffer, and 12 different concentrations of APH dilutions including 0 μM were obtained by two-fold serial dilution. 3 μL of cyclic peptide solution and 12 μL of APH solution of a specific concentration were added to each well of a 384-well ELISA plate. Incubate at room temperature, and use a multifunctional ELISA reader to detect the fluorescence anisotropy (A) value of each sample well.

[0109] Data were analyzed using GraphPad Prism 5 software, and the dissociation constant (K) was calculated by the nonlinear regression analysis formula of fluorescence anisotropy (A) and APH concentration. D ).

[0110]

[0111] The results of fluorescence polarization experiments showed that the 12 candidate cyclic peptide molecules of the present invention exhibited nanomolar or low micromolar affinity for the target protein APH. The test results are shown in Tables 1 and Figure 3 shown.

[0112] 8. Determination of the inhibitory effect of candidate cyclic peptide AR-L3 on target protein APH enzyme activity

[0113] In this example, the HPLC method was used to detect the ADP content of the reaction product, and then the inhibition of the candidate cyclic peptide AR-L3 on the target protein APH enzyme activity was determined.

[0114] In this experiment, the candidate peptide AR (ACSWPLCELLGRR-NH2) was synthesized and modified with L3 to obtain the cyclic peptide AR-L3.

[0115] The HPLC mobile phase consisted of 95% 0.02M KH2PO4 (pH=6.0) and 5% Methanol, the flow rate was 1 mL / min, the detection wavelength was set at 254 nm, and the injection volume was 10 μL.

[0116] ADP-Na2 standard curve drawing: Prepare 200mM ADP-Na2 standard solution with reaction buffer (50mM Tris, 40mM KCl, 10mM MgCl2, pH=7.5), and then dilute with reaction buffer to obtain ADP-Na2 standard solutions with a series of concentrations (32μM, 16μM, 8μM, 4μM, 2μM and 1μM). Perform HPLC determination on the ADP-Na2 standard solution samples with a series of concentrations, and draw the ADP-Na2 standard curve based on the measured peak area and concentration.

[0117] Determination of the effect of cyclic peptide on APH catalytic activity: Different concentrations of cyclic peptide AR-L3 were added to the APH enzymatic reaction system to determine the inhibitory effect of the cyclic peptide on the enzyme activity. In this study, a total of 7 different reaction systems were set up as shown below, including a positive control group (1), an experimental group (2-6) and a negative control group (7).

[0118]

[0119]

[0120] Take 20μL of the above 7 reaction samples at 0min and 60min in a 37℃ water bath, mix with 40μL chloroform to terminate the reaction, and vortex mix for 1min. Then, add 20μL or 60μL reaction buffer to the termination reaction solution at 0min and 60min, vortex mix for 1min, and let stand at room temperature for 2-3min. Centrifuge at 4℃, 12000×g for 5min, take 10μL of the supernatant for HPLC analysis to detect the ADP content in the reaction solution. Set up three parallels for each sample.

[0121] A standard curve was drawn based on the peak area of ​​5 concentrations of ADP-Na2 standard at 254 nm, with sample concentration as the horizontal coordinate x (μM) and peak area as the vertical coordinate y (mAU*s), and the linear regression equation y=5.3679x-3.4096 was obtained, R 2 =0.9991.

[0122] The results of measuring the ADP content in the reaction solution are as follows Figure 4 As shown, after 60 minutes of reaction, compared with the sample containing 0 μM AR-L3 in the reaction system, the content of reaction product ADP decreased when 0.31 μM, 0.63 μM, 1.25 μM, 2.50 μM and 5.00 μM AR-L3 were added, and the ADP content showed a downward trend with the increase of AR-L3 concentration in the reaction system. For example, when 0.31 μM, 1.25 μM and 2.50 μM AR-L3 were added, the enzyme activity of APH was reduced to about 60%, 50% and 30% of the sample containing 0 μM AR-L3 in the reaction system.

[0123] Example 2

[0124] APH is a common aminoglycoside antibiotic resistance enzyme, so the development of APH cyclic peptide inhibitors is a way to treat the corresponding drug-resistant bacteria. The 12 APH candidate cyclic peptide inhibitor molecules, after in vitro synthesis and modification, are mixed with the corresponding aminoglycoside antibiotics in different proportions and used in a specific way. They can be applied to the treatment of different drug-resistant bacteria and have good application prospects.

[0125] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An aminoglycoside phosphotransferase cyclic peptide inhibitor, characterized in that Selected from one of the polypeptides having the following amino acid sequence: (1)Ala-Cys-Ser-Trp-Pro-Leu-Cys-Glu-Leu-Leu; (2)Cys-Gly-Trp-Leu-Leu-Cys-Pro-Phe-Val; (3)Ile-Cys-Ala-Trp-Pro-Leu-Cys-Asn-Leu-Leu; (4) Cyclic peptides containing the conserved sequence Cys-X-Trp-X-Leu-Cys, where X is any amino acid; (5) A cyclic peptide containing the conserved sequence Cys-Pro-X-Tyr-Cys, wherein X is any amino acid.

2. The aminoglycoside phosphotransferase cyclic peptide inhibitor according to claim 1, characterized in that The aminoglycoside phosphotransferase cyclic peptide inhibitor is selected from one of A-L2, A-L3, A-L4, A-L7, C-L2, C-L3, C-L4, I-L2, I-L3, I-L4, I-L7 or Y-L5:

3. An isolated nucleic acid, characterized in that Encoding the aminoglycoside phosphotransferase cyclic peptide inhibitor according to claim 1 or 2.

4. A carrier, characterized in that The vector comprises the nucleic acid of claim 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the aminoglycoside phosphotransferase cyclic peptide inhibitor according to claim 1 or 2, and one or more pharmaceutically acceptable excipients and / or carriers.

6. Use of the aminoglycoside phosphotransferase cyclic peptide inhibitor according to claim 1 or 2 in the preparation of a therapeutic drug for aminoglycoside antibiotic-resistant bacteria.

7. The use according to claim 6, characterized in that The aminoglycoside phosphotransferase cyclic peptide inhibitor is used in the preparation of a drug for treating drug-resistant bacteria associated with APH expression.

8. Use of the aminoglycoside phosphotransferase cyclic peptide inhibitor according to claim 1 or 2 in the preparation of a therapeutic drug for treating diseases related to the expression of protein kinases with similar sequences to APH.

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