Aminoglycoside phosphotransferase loop peptide inhibitors and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to aminoglycoside phosphotransferase cyclic peptide inhibitors and uses thereof. BACKGROUND

[0002] The problem of antibiotic resistance is one of the major challenges in the field of global public health. Aminoglycoside resistance is mainly caused by the catalytic action of aminoglycoside-modifying enzymes (AMEs). These enzymes can make antibiotics lose their antibacterial activity through chemical modification. Among them, aminoglycoside phosphotransferases (APHs) are a typical AME that can transfer the phosphate group of ATP to the hydroxyl group on the antibiotic molecule, resulting in its inactivation, such as APH(3')-I, etc.

[0003] To cope with the increasingly serious problem of bacterial resistance, in addition to reducing antibiotic use, developing new antibiotics and strengthening monitoring of drug-resistant bacteria, designing new inhibitors targeting AMEs is an important research strategy. An ideal AME inhibitor 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 their proliferation. However, despite more than 40 years of research on AME inhibitors, there have been no reports of cyclic peptide AME inhibitors, and no AME-targeted inhibitors have been applied to the clinic.

[0004] Aminoglycoside phosphotransferases (APH) are a well-studied AME, and due to their structural similarity to 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 these inhibitors in complex with APH, and found that Pyrazolopyrimidine derivatives can significantly inhibit the activity of APH(3')-Ia, restoring the antibacterial effect of antibiotics on drug-resistant E. coli. However, to achieve clinical application, these inhibitors still need to be further optimized to retain inhibitory activity while avoiding affecting normal physiological functions of the host.

[0005] Polypeptide drugs are attracting much attention in drug research and development due to their ease of synthesis, significant pharmacological activity, and high-efficiency interaction with protein targets. Compared with linear polypeptides, cyclic peptides have higher affinity, specificity, stability, and cell membrane permeability. The diverse cyclization methods of cyclic peptides, such as disulfide bonds, amide bonds, and other covalent bonds, enable them to exhibit unique advantages in drug research and development. Currently, more than 40 cyclic peptide drugs have been approved for marketing by the FDA, including cyclosporin, desmopressin, and anidulafungin.

[0006] Despite the progress made in the field of cyclic peptide drugs, there is still a gap in the development of cyclic peptide inhibitors targeting aminoglycoside phosphotransferase (APH). Developing cyclic peptide molecules with high affinity for APH and high-efficiency 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

[0007] Developing inhibitors of drug-resistant enzymes is a method to address the problem of bacterial drug resistance. The present application provides aminoglycoside phosphotransferase cyclic peptide inhibitors and their uses. The present application screens a series of cyclic peptide inhibitors targeting aminoglycoside phosphotransferase, laying a molecular foundation for the development and application of aminoglycoside phosphotransferase cyclic peptide inhibitors.

[0008] The object of the present application can be achieved by the following technical solutions:

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

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

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

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

[0013] (4) a cyclic peptide containing the conserved sequence Cys-X-Trp-X-Leu-Cys, wherein 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 application, the aminoglycoside phosphotransferase cyclic peptide inhibitor is selected from one of the following: 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 application all contain two cysteines. The side chain sulfhydryl groups of the cysteines in the polypeptide undergo affinity substitution reaction with the active halogen Br on the chemical cross-linking agent, thereby obtaining the cyclic peptide by modification. 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 application and their binding affinity to APH are shown in Table 1.

[0020] Table 1 Sequence and binding affinity of APH candidate cyclic peptide inhibitors

[0021]

[0022] The present application further provides a method for obtaining cyclic peptide molecules that specifically target APH. The method can be obtained by phage display technology. First, the target protein APH is purified in vitro, and candidate cyclic peptides of the target protein are screened from a phage display library of 1010 11 The screening process includes the steps of "preparation and blocking of phage cyclic peptide library - binding and blocking of target protein to magnetic beads - phage display screening". Multiple rounds of screening are performed by increasing the screening pressure in different screening rounds, and the phage titer after each round of screening is measured by the method of plaque count. The specific binding phage enriched in each round of screening is sequenced using the Illumina next-generation sequencing platform, and the sequences and abundance of the target APH specific binding peptides are obtained by processing the next-generation sequencing data with Matlab scripts.

[0023] The present application further provides a modification and purification method of the specific APH-targeting cyclic peptide molecule. In one embodiment of the present application, the modification and purification method of the specific APH-targeting cyclic peptide molecule is as follows: the modification reaction system is 100 mM NH4HCO3(pH 8.0), 50% ACN, 0.5 mM polypeptide and 1 mM chemical cross-linking agent, and the reaction is carried out at 30°C for 1 h. 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 after verifying the molecular weight by mass spectrometry, it is freeze-dried.

[0024] The present application uses fluorescence polarization technology to determine the binding affinity of the cyclic peptide molecule to the target protein APH. It is determined that the affinity of the cyclic peptide of the present application to APH is in the low micromolar level, which lays a foundation for the development of cyclic peptide inhibitor molecules targeting APH.

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

[0026] The present application further provides a vector comprising the nucleic acid.

[0027] The present application further provides a pharmaceutical composition comprising the aminoglycoside phosphotransferase cyclic peptide inhibitor (specific APH-targeting cyclic peptide molecule) and one or more pharmaceutically acceptable excipients and / or carriers.

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

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

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

[0031] The application provides a cyclic peptide molecule specifically targeting APH and use thereof. Aminoglycoside phosphotransferase APH is an aminoglycoside antibiotic resistance enzyme, and developing resistance enzyme inhibitors is an important way to treat drug-resistant bacteria. The application uses phage display technology widely used in polypeptide or antibody drug development to screen APH protein for multiple rounds, and obtains candidate cyclic peptide molecule sequences specifically binding to APH through second-generation sequencing of enriched sequences. The cyclic peptide molecule contains two cysteines, is modified into a ring through a specific chemical crosslinking agent, and has a low micromolar affinity for the target protein APH as determined by fluorescence polarization technology. It is found through experiments that the cyclic peptide shows an inhibitory effect on the enzyme activity of APH, and the cyclic peptide in the study lays a foundation for the development of APH targeting inhibitors.

[0032] (1) Compared with traditional antibodies or small molecule inhibitors, the cyclic peptide molecule of the application has the advantages of high affinity, strong targeting, small volume, easy synthesis and modification, low cost, simple operation, flexible drug delivery route and the like.

[0033] (2) The cyclic peptide molecule of the application is reported for the first time at home and abroad, and has innovation.

[0034] (3) The 12 cyclic peptides specifically targeting APH provided by the application can be applied to the treatment of aminoglycoside antibiotic resistant bacteria, and have a potential clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced below, so that those skilled in the art can better understand the application and implement it.

[0036] Figure 1 : The enrichment degree of the candidate cyclic peptide specifically targeting APH in the screening process is determined according to the phage titer.

[0037] Figure 2 : The polypeptide sequences and abundance of the polypeptides specifically targeting APH obtained through three rounds of phage display screening under the conditions of four crosslinking agents.

[0038] Figure 3 : The experimental results of using fluorescence polarization technology to characterize the binding affinity of 12 cyclic peptide molecules to the target protein APH.

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

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

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

[0042]

[0043]

[0044] The main reagent formulations of the following examples of the present application are as follows:

[0045] 1, 10 mg / mL tetracycline (10 mL): 100 mg tetracycline powder was dissolved with 50% ethanol to 10 mL. After sterilization by 0.22 μm filter membrane, it was divided into 1.5 mL centrifuge tubes and stored at -20°C for long-term preservation.

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

[0047] 3, Buffer N (1 L): 200 g polyethylene glycol 6000, 146.1 g NaCl, distilled water to 1 L, 4°C for long-term preservation.

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

[0049] 5, 20 mM TCEP (5 mL): 28.7 mg TCEP, distilled water to 5 mL, -20°C for long-term preservation.

[0050] 6, Preparation method of 10 mM chemical cross-linking agent solution: 1,4- bis(bromomethyl) benzene (abbreviation: Linker 1), 1,3-bis(bromomethyl) benzene (Linker 2), 2,6-bis(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) cross-linking agent solutions were prepared with acetonitrile solution, and 2,6-bis(bromomethyl) naphthalene (Linker 6) cross-linking agent solution was 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, distilled water to 1 L.

[0052] 8. Wash Buffer 2 (10 mL): 10 mL Wash Buffer 1, 100 mg BSA and 10 μL Tween 20.

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

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

[0055] Table 3 List of primers used for preparation of NGS library in the examples of the present application

[0056]

[0057]

[0058] Example 1

[0059] Screening and identification of phage display random cyclic peptide library targeting APH:

[0060] According to the screening procedures of "Preparation of phage cyclic peptide library and blocking-target protein binding with magnetic beads and blocking-phage display screening", a total of 3 rounds of screening were performed. The protein amount was gradually reduced to increase the screening stringency, and Streptavidin magnetic beads and Neutravidin magnetic beads were alternately used in each round of screening to reduce non-specific binding in the screening process. The specific conditions are shown in Table 4. Among them, the Neutravidin magnetic beads were prepared according to the instructions (2 mg Neutravidin protein was incubated with 3350 μL of 30 mg / mL Dynabeads M-280 Tosylactivated). TM M-280 Tosylactivated).

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

[0062]

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

[0064] 1) 1 mL of E. coli TG1 glycerol stock containing the phage library described in Table 2 (a total of 11 libraries) was inoculated into 250 mL of 2YT liquid medium (containing 10 μg / mL tetracycline), 30°C, 200 rpm, and cultured for 9-15 h;

[0065] 2) All the culture solutions were centrifuged at 4°C, 12000 x g, 20 min;

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

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

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

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

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

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

[0072] 9) Add 0.5 mL of the corresponding chemical cross-linking agent (100 mM Linker) to each 4.5 mL of phage solution, and place 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, centrifuge at 4°C, 4500 rpm, 15 min;

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

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

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

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

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

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

[0080] 3. Phage display screening

[0081] 1) Respectively mix 5 mL phage solution obtained in step 1 with 50 μL target protein (with magnetic beads) sample obtained in step 2, and place in a rotary mixer (10 rpm, 1 h) for binding reaction;

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

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

[0084] 4) Transfer the resuspension into 5 mL of E. coli TG1 bacterial solution (OD 600 = 0.5-0.8), and infect at 37 °C for 30 min;

[0085] 5) Collect the bacteria at 4500 rpm for 10 min, resuspend in 500 μL of 2YT medium, take 10 μL of the bacterial solution and add to 90 μL of 2YT medium, and then dilute by 10 times gradient 7 times in sequence, take 5 μL of each gradient dilution and plate on 2YT solid medium (containing 10 μg / mL tetracycline) plate, and culture overnight at 37 °C. The remaining bacterial solution is plated on 2YT medium plate (containing 10 μg / mL tetracycline).

[0086] 4. Multiple rounds of phage display screening

[0087] After completing the first round of phage display screening, 2-3 rounds of screening are required to improve the abundance of candidate cyclic peptides bound to the target protein. On the one hand, the main experimental steps such as cyclic peptide library preparation and blocking, target protein immobilization and blocking, and screening are repeated, and on the other hand, the screening conditions are gradually tightened, such as reducing the amount of target protein, and alternately using streptavidin magnetic beads and neutravidin magnetic beads (to reduce non-specific binding in the screening process) in multiple rounds of screening.

[0088] 5. Sequencing and analysis of enriched samples

[0089] The recombinant phages obtained after multiple rounds of screening are subjected to two rounds of PCR. The experimental purpose of the first round of PCR is to add specific barcode sequences to both sides of each sample and introduce an index complementary sequence, and the experimental purpose of the second round of PCR is to obtain consistent index sequences for the samples. The PCR product is detected by electrophoresis on a 2.5% agarose gel. The PCR samples after two rounds of reaction are sent to Suzhou Jinyizhi Biological Technology Co., Ltd. for sequencing, and the Illumina Novaseq 2x150bp sequencing platform is used.

[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: Sample enriched in each round of screening and primer pair control table

[0093]

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

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

[0096] 2) Second round of PCR:

[0097] The product of the first step PCR was mixed according to the brightness of the band in proportion as the template for the second step PCR. 2x NG Master Mix was used for PCR reaction.

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

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

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

[0101] 3) Next-generation sequencing was completed by Jinweizhi Biotechnology Co., Ltd. on the Illumina Novaseq 2x150bp sequencing platform. The sequencing process mainly includes construction of sequencing library, bridge PCR amplification and denaturation, and sequencing. After sequencing, about 100 million pieces of sequencing result data with a length of about 240 bp 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 optimized for data analysis. The content of NGS sequencing data analysis mainly includes sorting and sequence translation of data according to specific primer sequences, clustering analysis of polypeptide sequences on this basis to obtain more accurate and effective candidate polypeptide sequences, and enrichment results and abundance of polypeptide sequences enriched under Linker 2, Linker 3, Linker 4 and Linker 5 conditions as shown in Table 1. Figure 2

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

[0105] Two linear polypeptide sequences containing two cysteines described in Table 1 were ordered from a polypeptide synthesis company. Each 0.5 mg of polypeptide 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 polypeptide and 1 mM chemical crosslinking 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 120 EC-C18(4.6x150mm, 4μm)), and the molecular weight of the purified product was verified by Shimadzu UPLC-MS (chromatographic column Shim-pack GISTC18(2.1x50mm, 2μm)), and the correct cyclic peptide was vacuum freeze-dried.

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

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

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

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

[0110] ​

[0111] The fluorescence polarization method experiment results show that the 12 candidate cyclic peptide molecules in the application exhibit nanomolar or low micromolar affinity to the target protein APH, and the test results are shown in Table 1 and Figure 3

[0112] 8. Determination of the inhibition of the target protein APH enzyme activity by the candidate cyclic peptide AR-L3

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

[0114] In this experiment, the candidate polypeptide AR (ACSWPLCELLGRR-NH2) is synthesized, and after L3 modification, the cyclic peptide AR-L3 is obtained.

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

[0116] ADP-Na2 standard curve drawing: 200 mM ADP-Na2 standard solution is prepared with Reaction buffer (50 mM Tris, 40 mM KCl, 10 mM MgCl2, pH = 7.5), and then a series of concentrations (32 μM, 16 μM, 8 μM, 4 μM, 2 μM and 1 μM) of ADP-Na2 standard solution are obtained by dilution with reaction buffer. The HPLC determination is performed on the series of concentration ADP-Na2 standard solution samples, and the ADP-Na2 standard curve is drawn according to 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 are added to the APH enzymatic reaction system to determine the inhibition of the enzyme activity by the cyclic peptide. In this study, 7 different reaction systems are set as shown below, including the positive control group (1), the experimental groups (2-6) and the negative control group (7).

[0118]

[0119]

[0120] ​The above 7 reaction samples were mixed with 40 μL chloroform to terminate the reaction at 37°C water bath for 0 min and 60 min, respectively, and vortexed for 1 min. Then, 20 μL or 60 μL Reaction buffer was added to the terminated reaction liquid at 0 min and 60 min, respectively, vortexed for 1 min, and left at room temperature for 2-3 min. Centrifugation was performed at 4°C, 12000 x g for 5 min, and 10 μL supernatant was taken for HPLC analysis to detect the ADP content in the reaction liquid. Three parallel samples were set for each sample.

[0121] A standard curve was drawn according to 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), to obtain the linear regression equation y = 5.3679x-3.4096, R 2 = 0.9991.

[0122] The results of measuring the ADP content in the reaction liquid are shown in Table 1. Figure 4 Compared with the sample containing 0 μM AR-L3 in the reaction system, when 0.31 μM, 0.63 μM, 1.25 μM, 2.50 μM and 5.00 μM AR-L3 was added, the content of the reaction product ADP was reduced, and with the increase of AR-L3 concentration in the reaction system, the ADP content showed a downward trend, for example, when 0.31 μM, 1.25 μM and 2.50 μM AR-L3 was 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. After in vitro synthesis and modification, 12 APH candidate cyclic peptide inhibitor molecules were mixed with corresponding aminoglycoside antibiotics in different proportions, and could be used for the treatment of different drug-resistant bacteria by specific methods, which had good application prospect.

[0125] The above description of the embodiments is to facilitate the understanding and use of the invention by those of ordinary skill in the art. 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 labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A cyclic peptide binding to an aminoglycoside phosphotransferase, characterized in that, The structure of the cyclic peptide is as follows: 。 2. An isolated nucleic acid, comprising, The nucleic acid of claim 2.

3. A vector, characterized in that, The vector comprises the nucleic acid of claim 2.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the cyclic peptide of claim 1, and one or more pharmaceutically acceptable excipients and / or carriers.

Images