Klebsiella pneumoniae LpxC enzyme inhibitor binding site determination method and application
By constructing human and animal-derived Klebsiella pneumoniae LpxC enzyme models, the binding site was determined and 5-methyl-3-hexen-2-one was screened as an LpxC enzyme inhibitor, which solved the problem of low screening efficiency in the existing technology and achieved effective inhibition of Klebsiella pneumoniae, especially when used in combination with polymyxin B.
Patent Information
- Application Number
- CN202511419032.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-02-06
AI Technical Summary
Existing technologies struggle to effectively screen for LpxC enzyme inhibitors suitable for Klebsiella pneumoniae, especially considering the combined challenges of drug safety and inhibitory efficacy. Traditional methods suffer from low screening efficiency and poor inhibitory effects.
By constructing a homology model based on human and animal Klebsiella pneumoniae LpxC enzyme, the binding site was determined. Using high-throughput computer screening of the ZINC15 natural compound database, combined with ADMET and drug-likeness screening criteria, 5-methyl-3-hexen-2-one was screened as an LpxC enzyme inhibitor, especially when used in combination with the antibacterial drug polymyxin B to enhance the inhibitory effect.
This study improved the applicability of screening LpxC enzyme inhibitors. The selected compounds, such as 5-methyl-3-hexen-2-one, can significantly inhibit Klebsiella pneumoniae, especially when used in combination with polymyxin B, which shows better antibacterial effect and provides new ideas for drug development.
Smart Images
Figure CN121483358A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of drug screening and new use of drugs, and particularly relates to a method for determining a binding site of a Klebsiella pneumoniae LpxC enzyme inhibitor and application thereof. BACKGROUND
[0002] In recent years, the widespread use and abuse of antibiotics has made the problem of bacterial drug resistance more and more serious, especially the emergence of multi-drug resistant pathogenic bacteria, which has further reduced the scope of use of antibiotics. This phenomenon has threatened human health and attracted the attention of the World Health Organization (WHO) and governments around the world. Therefore, it is urgent to find new antibacterial drugs and means to curb the emergence of multi-drug resistant pathogenic bacteria.
[0003] Klebsiella pneumoniae (KP) is an important acquired pathogen that can cause a variety of serious infections, including pneumonia, urinary tract infection, sepsis, wound infection, and meningitis. The enzyme encoded by the LpxC gene is a key enzyme in the synthesis of the bacterial cell wall, and thus becomes an important target for the development of antibacterial drugs. LpxC enzyme inhibitors can exert bactericidal effects by blocking the synthesis of the bacterial cell wall, and have potential value for the treatment of infections caused by drug-resistant strains.
[0004] Gram-negative bacteria have a unique outer membrane (OM) structure, which is a natural physical barrier that can hinder drug penetration into bacteria and activate efflux pumps, which is an important reason for the poor efficacy of many antibiotics against Gram-negative bacteria. The structure is composed of three layers, with lipopolysaccharide (LPS) on the outer layer, a phospholipid layer in the middle layer, and a lipoprotein layer on the inner layer. Lipopolysaccharide is a special substance that distinguishes Gram-negative bacteria from positive bacteria, and can stabilize the outer membrane structure of Gram-negative bacteria. Lipopolysaccharide is composed of three main parts: O-antigen, core polysaccharide, and lipid A (LA), among which lipid A plays an important role in the correct assembly of lipopolysaccharide (LPS) and anchoring, and is also an important component of bacteria to protect against external factors (such as antibiotics and detergents), and is a powerful endotoxin that can trigger a very strong immune response in the host, even leading to death (septic shock), which is an important cause of Gram-negative bacterial infection. Therefore, lipid A plays an important role in the survival of Gram-negative bacteria, and by inhibiting the synthesis of lipid A, the formation of LPS can be prevented, thereby weakening the pathogenic ability of bacteria.
[0005] There are 9 enzymes involved in the synthesis of LA, and the deacetylation reaction catalyzed by the deacetylase (LpxC) encoded by the LpxC gene is the first key step in the synthesis pathway, and effective inhibition of LpxC can indirectly interfere with the synthesis of LPS, ultimately leading to the death of the bacterial cell. LpxC as an ideal antibacterial drug target enzyme, on the one hand, it has no homologous protein sequence in humans and other mammals, on the other hand, its inhibitors have the advantages of low off-target and low toxicity.
[0006] So far, LpxC as one of the most promising targets for developing anti-Gram-negative bacterial drugs, many types of inhibitors and derivatives have been reported. Some hydroxamic acid-containing compounds have shown good antibacterial activity after continuous structural optimization, thus becoming classic inhibitors. In particular, among the sulfonamide hydroxamic acid derivatives reported in 2002, BB-78485 stands out with its remarkable antibacterial activity, representing an important development in this class of drugs. In 2005, a series of new hydroxamic acid inhibitors were reported, among which CHIR-090 was recognized as the best inhibitor lead with the best activity reported in the literature. CHIR-090 is characterized by its unique binding mode: it slowly and tightly binds to the LpxC enzyme through a two-step reaction, showing time dependence and a long drug half-life. More importantly, it has the widest antibacterial spectrum among existing inhibitors, and can efficiently inhibit LpxC enzyme, with antibacterial potency even comparable to that of ciprofloxacin and tobramycin. Therefore, CHIR-090 is considered to be the most successful LpxC enzyme inhibitor lead compound structure so far, but its development was terminated due to adverse reactions such as injection site inflammation in clinical trials, which provides important experience and lessons for the design and development of subsequent LpxC enzyme inhibitors, especially in terms of safety evaluation and structural optimization of drugs.
[0007] Therefore, there is still a need to further screen effective LpxC enzyme inhibitors for inhibiting Klebsiella pneumoniae. SUMMARY
[0008] The purpose of the application is to provide a method for determining the binding site of Klebsiella pneumoniae LpxC enzyme inhibitors and its application.
[0009] Technical scheme: In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows:
[0010] In a first aspect, the present application provides a method for determining the binding site of Klebsiella pneumoniae LpxC enzyme inhibitors, comprising the following steps:
[0011] 1) Selecting different homology levels of Klebsiella pneumoniae strains, using SWISS MODE website to build homology model of strain gene sequence and optimizing with GalaxyRefine, obtaining LpxC enzyme protein model;
[0012] 2) Verifying the binding sites obtained from software and literature, evaluating the advantages and disadvantages of the binding pockets according to the obtained 2D structure diagram, obtaining LpxC enzyme protein binding pocket.
[0013] As a specific embodiment, the selection of different homology levels of Klebsiella pneumoniae strains is to select one strain from each of the strains showing different homology levels as a representative, including human and animal sources of Klebsiella pneumoniae strains.
[0014] As a specific embodiment, in step 2), the determination of the LpxC enzyme protein binding pocket is to verify the advantages and disadvantages of these pockets with more mature hydroxyl amide compounds according to the literature and the binding sites automatically generated by software.
[0015] As a specific embodiment, in step 2), four LpxC enzyme protein binding pockets are determined, and the key amino acids are as follows:
[0016] Binding pocket (1): L143, A160, P161, P192, P194, L239, A242, L262, H265;
[0017] Binding pocket (2): L18, C63, E78, H79, T191, F192, M195, I198, F212, V217, H238, K239, D242, H265;
[0018] Binding pocket (3): E78, H79, T191, H238, D242, D246, H265;
[0019] Binding pocket (4): L18, H19, M61, L62, C63, T76, G78, H79, I103, G106, A190, T191, P192, G193, P194, M195, A196, A197, I198, G199, T200, L201, G202, L206, C207, L208, G209, G210, S211, P212, C214, A215, I216, V217, V218.
[0020] The abbreviations in the above binding pockets are represented by letters representing amino acids and numbers representing the positions of amino acids, such as binding site L143, which represents Leucine Leu at position 143 in the amino acid sequence of LpxC enzyme protein as a binding site.
[0021] In a second aspect, the present application provides a method for screening a Klebsiella pneumoniae LpxC enzyme inhibitor, comprising the following steps:
[0022] 1) Selecting Klebsiella pneumoniae strains with different homology levels, using SWISS MODE website to perform homology modeling on the strain gene sequence and using GalaxyRefine to optimize, to obtain a LpxC enzyme protein model;
[0023] 2) Simultaneously verifying the binding sites obtained from software and literature, evaluating the advantages and disadvantages of the binding pocket according to the obtained 2D structure diagram, to obtain a LpxC enzyme protein binding pocket;
[0024] 3) Selecting a classic ZINC15 natural compound database for high-throughput screening against the determined LpxC enzyme protein model and LpxC enzyme protein binding pocket, to obtain a preliminary screening small molecule;
[0025] 4) Performing ADMET and drug-like property screening on the preliminary screening small molecule, to finally obtain a target inhibitor.
[0026] As a specific embodiment, in step 3), the ZINC15 natural compound database screening standard is Affinity≤-7 kcal / mol.
[0027] As a specific embodiment, in step 4):
[0028] The ADMET screening standard is: aqueous solubility is 4 (-2.0<log(Sw)<0.0=“”); blood-brain barrier penetration is 1 (0≤logBB<0.7); intestinal absorption is 1; cannot have liver toxicity; cannot bind to plasma proteins; cannot inhibit CYP2D6; cannot cause skin irritation;
[0029] The drug-like property screening standard is based on the five rules of Lipinski, which are: no more than 5 hydrogen bond donors; no more than 10 hydrogen bond acceptors; no more than 5 lipophilic water partition coefficients (LogP); molecular weight is below 500; and no more than 10 rotatable bonds.
[0030] In a third aspect, the present application provides use of the LpxC enzyme inhibitor obtained by the screening method in the preparation of a drug for inhibiting Klebsiella pneumoniae.
[0031] In a fourth aspect, the present application provides use of 5-methyl-3-hexen-2-one in the preparation of a drug for inhibiting Klebsiella pneumoniae.
[0032] In a fifth aspect, the present application provides use of 5-methyl-3-hexen-2-one in combination with an antibacterial drug in the preparation of a drug for inhibiting Klebsiella pneumoniae.
[0033] Preferably, the antibacterial drug is polymyxin B.
[0034] Beneficial effects: Compared with the prior art, the present application is based on the LpxC enzyme sequences of homologous human and animal sources of Klebsiella pneumoniae to construct an LpxC enzyme protein model, then determine the LpxC enzyme protein binding site, and finally use a computer to screen out an inhibitor suitable for the structure of the LpxC enzyme protein by high-throughput simulation. This method is more suitable for LpxC enzyme and provides a new idea for the screening of LpxC enzyme inhibitors. And the screened 5-methyl-3-hexen-2-one, as an LpxC enzyme inhibitor, can effectively inhibit Klebsiella pneumoniae, especially when combined with the antibacterial drug polymyxin B, the inhibitory effect is better. BRIEF DESCRIPTION OF DRAWINGS
[0035] Figure 1 Figure 8 is an SNP phylogenetic tree of 1807 strains of Klebsiella pneumoniae, the outer circle is the strain source, divided into human and animal sources; the inner circle is the LpxC gene of different homology.
[0036] Figure 2 Figure 9 is the LpxC protein model obtained after final modification and optimization.
[0037] Figure 3 Figure 10 is the LpxC protein model Ramachandran plot, the allowed zone amino acid number ratio is 100%, the disallowed zone amino acid ratio is 0%, the model <5%, meets the evaluation requirements, the core area (red) residue is 90.7%, and the model >90% is considered to be an ideal model.
[0038] Figure 4 Figure 11 is the molecular docking result, wherein ABC is the binding pocket (1) and the docking results of CHIR-90 and BB-78485 molecules with the binding pocket (1); D-I is the binding pocket (2), (3), (4) and the docking results of CHIR-90 molecules with the binding pocket (2), (3), (4), respectively.
[0039] Figure 5 Figure 12 is the ADMET screening result interval, the basket: ADMET_AlogP98; the red circle: Absorption-95; the green circle: Absorption-99; the purple circle: BBB-95; the light blue circle: BBB-999.
[0040] Figure 6 Figure 13 is the molecular docking visualization of LpxC and the screened compound ZINC1765531, the compound has hydrogen bond force with the amino acids ASP242 and HIS238, and the distances are
[0041] Figure 7 Figure 14 is a minimum inhibitory concentration (MIC) heat map of 14 strains of Klebsiella pneumoniae with different antibacterial drugs.
[0042] Figure 8 FIC index for checkerboard method of drug susceptibility screening.
[0043] Figure 9 Time-kill curve of polymyxin B and T combination against 19K-1068.
[0044] Figure 10 Time-kill curve of polymyxin B and T combination against 19K-979. DETAILED DESCRIPTION
[0045] The present application is further illustrated by the following examples. These examples are purely illustrative and are not intended to limit the present application. The present application is further described below in conjunction with the accompanying drawings and examples.
[0046] The following strains were used in the experiments of the examples: Klebsiella pneumoniae 19K-1068 (ST48 type), 19K-69 (ST11 type), 20K-106 (ST410 type), 19K-202 (ST131 type), 19K-1130 (ST16 type), 20K-449 (ST659 type), 19K-979 (ST23 type), 19K-845 (ST11 type), 19K-271 (ST48 type), 19K-847 (ST11 type) and 768 (ST31 type), which are all conventional types of bacteria in the art and were isolated and preserved in the laboratory of the applicant; and ATCC 700603, which is a commonly used quality control strain in the art.
[0047] Example 1 Screening of LpxC enzyme inhibitors of Klebsiella pneumoniae
[0048] 1. Alignment of LpxC gene sequences
[0049] In the process of exploring LpxC enzyme inhibitors of Klebsiella pneumoniae, the present study faced the challenge of genetic sequence diversity. In order to overcome this difficulty, the present study selected 1748 strains of human-derived Klebsiella pneumoniae and 59 strains of animal-derived Klebsiella pneumoniae, as shown in Table 1. Figure 1To ensure that the LpxC enzyme gene sequences of the selected strains are sufficiently representative and diverse, the strains showing different levels of homology were selected as representatives, and finally 39 strains of Klebsiella pneumoniae of human origin and 12 strains of animal origin were screened. The gene sequences of these strains were analyzed in detail using SnapeGene software. This analysis not only revealed the conserved regions in the sequence, but also identified the mutation regions, providing a solid theoretical basis for determining the binding site in virtual screening. The results of this study showed that the LpxC enzyme amino acid sequences of Klebsiella pneumoniae of human and animal origin have up to 96% similarity. This finding indicates that although there are certain differences in the sequence, the key active regions are largely conserved.
[0050] 2. Homology modeling
[0051] Through second-generation sequencing, gene search obtained LpxC enzyme protein sequence, due to different degrees of gene mutation, LpxC enzyme protein sequence is not the same, this study with and Escherichia coli MG1655 (derived from W1485 strain, is a derivative strain of K-12, is a relatively close to "WT-wild type" of Escherichia coli engineering strain after less modification.) 100% homologous strain 19K-1058 sequence as a template homology modeling with SWISS MODEL website, get 20 LpxC protein model, its structure is similar, the main difference is the difference in spatial conformation, put the obtained model into GalaxyRefine for pretreatment and modification of wrong conformation, optimize the processed model, according to the difference in spatial conformation, respectively optimized, finally according to the PDFTotal Energy, DOPE score value screening. Among them, the PDF function defines the geometric properties such as bond length, bond angle, dihedral angle and other properties in the protein structure, which reflects the pros and cons of the model. The higher the PDF Total Energy of the model, the better the model is optimized under the homology constraint, and the higher the reliability of the model. DOPE score is the basis for measuring the quality of the model, the lower the score, the more reliable the quality of the model. According to the score, LpxC.M0018 is finally selected as the LpxC protein model, as shown in the accompanying Figure 2 .
[0052] In the process of verifying the accuracy and reliability of the LpxC protein model, this study uses the Ramachandran plot analysis to determine whether the model is available, as shown in the accompanying Figure 3The allowed region amino acid percentage reached 100% in the analysis of this study, which means that all the amino acid residues are located within the region considered biochemically reasonable. In addition, the disallowed region amino acid percentage is 0%, which further confirms the high quality of the model. The core region (usually represented in red) has an amino acid residue percentage of 90.7%, which is >90% identified as the ideal model standard, indicating that the model of this study has a high degree of credibility and accuracy. Finally, the LpxC protein model used for molecular docking was obtained.
[0053] 3. Determining the binding pocket
[0054] In the molecular docking study of LpxC enzyme inhibitors, this study first obtained a series of key-acting amino acid sites from the literature and confirmed that these sites are located in the conserved sequence. This step is crucial because it ensures that the amino acid sites selected in this study are highly conserved in different strains, thereby increasing the potential applicability of the inhibitors screened. The binding pocket position was determined by selecting key-acting amino acids in the molecular docking software AutoDock Vina. This study paid special attention to hydroxyl amide compounds because they have been proven to be effective LpxC enzyme inhibitors. These compounds inhibit the synthesis of LA, and thus inhibit the synthesis of LPS, reducing the toxicity of bacteria. In this study, through molecular docking, the binding affinity and mode of these compounds with the LpxC protein model were evaluated. Finally, based on the binding energy and hydrogen bonds in the docking results, the effect of the selected binding pocket was evaluated. The lower the binding energy, the stronger the interaction between the ligand and the receptor, and the presence of hydrogen bonds further confirms the specificity of the interaction.
[0055] In the molecular docking study of LpxC enzyme inhibitors, this study first determined a set of key-acting amino acid sites, including LYS143, ASP160, PHE161, PHE192, PHE194, LYS239, ASP242, LYS262, HIS265. The determination of these sites is based on their location in the conserved sequence and their role in forming the binding pocket. Through these sites, this study obtained the model of the binding pocket, as shown in Figure 6 This study selected hydroxyl amide compounds as potential inhibitor small molecules for molecular docking because this class of compounds has the ability to bind and inhibit LpxC enzyme in vitro with nanomolar level potency, as well as the advantage of exhibiting antibacterial activity in vivo. Representative compounds include CHIR-90 and BB-78485.
[0056]
[0057] In the molecular docking software AutoDock Vina, the present study determined the location of the binding pocket according to these key amino acid sites, and performed molecular docking experiments. The docking results showed that CHIR-90 and BB-78485 formed 4 and 5 carbon-hydrogen bonds, respectively, with the LpxC protein, as shown in Figs. Figure 4 B and C, indicating a relatively firm binding. To further verify the effect of the selected binding pocket, the present study also docked another three binding pockets, and the results showed that the number of carbon-hydrogen bonds in these pockets was four, which was slightly inferior to the first binding pocket, as shown in Figs. Figure 4 D-I. Therefore, the present study selected the first binding pocket as the site for molecular docking, which will provide an important reference for the subsequent drug design and screening of the present study.
[0058] 4. Large-scale small molecule docking screening of ZINC15 database
[0059] The classic ZINC15 natural product database (containing 270549 small molecules) was selected for docking screening, and 123876 small molecules were obtained through preliminary screening. The small molecules with poor scores were screened out by the confidence interval score, as shown in Fig. Figure 5 The docking score of Vina is Affinity, which represents the strength of the binding force. The smaller the value, the stronger the binding force. According to Affinity≤-7 kcal / mol, the binding force is strong, and 102488 small molecules were obtained for the next step of screening.
[0060] 5. ADMET and drug-likeness screening
[0061] To evaluate the drugability of the screened compounds, the ADMET module of the GHDDI-AIDD platform was selected for screening, including water solubility, blood-brain barrier penetration, CYP2D6 binding, hepatotoxicity, intestinal absorption, and plasma protein binding. Drug-likeness screening was performed using Lipinski's "Rule of Five" (RO5), which has the following rules: no more than 5 hydrogen bond donors; no more than 10 hydrogen bond acceptors; LogP no more than 5; molecular weight below 500; and no more than 10 rotatable bonds.
[0062] After ADME screening, 260 compounds were obtained from the 102488 compounds obtained by molecular docking. After drug-likeness screening, 6 compounds ZINC1765531, ZINC5355869, ZINC5193911, ZINC1841420, ZINC100018180, and ZINC36294388 were finally screened out, and their structural formulas are as follows:
[0063]
[0064] Among them, ZINC5355869, ZINC5193911, and ZINC100018180 have relatively mild drug properties and high safety. ZINC5193911 and ZINC100018180 are readily available. Considering cost, ZINC100018180, namely 5-methyl-3-hexen-2-one, was ultimately selected as a potential inhibitor for subsequent antibacterial activity evaluation and named T.
[0065] Table 1 Small Molecule Scoring
[0066]
[0067] 6. Molecular docking visualization
[0068] Visualizing molecular docking results is a crucial step in drug design, providing a direct understanding of the interactions between ligands (small molecule compounds) and receptors (target proteins). Taking compound ZINC1765531 as an example, this small molecule exhibits hydrogen bonding with amino acids HIS238 and ASP242, as shown in the attached diagram. Figure 6 As shown, this indicates that it may have formed a stable interaction with the active site of the enzyme.
[0069] Example 2: Study on the inhibition of Klebsiella pneumoniae by small molecule compound T
[0070] 1. Drug sensitivity test
[0071] (1) Stranded strains 19K-1068, 19K-826, 19K-69, 20K-106, 19K-202, 20K-196, 19K-1130, 20K-449, 19K-979, 19K-845, 19K-271, 19K-847, 768 and 700603 were incubated on MH agar plates at 37°C for 10 h, and then single strains were incubated in 1 mL of fresh MH broth at 37°C for 6 h.
[0072] (2) Take 100 μL of bacterial solution and add it to 900 μL of fresh MH broth. Mix well, then take 20 μL of bacterial solution and dilute it 1,000 times in a 90 mm plate containing 20 mL of MH broth. Use an eight-pipette to mix well.
[0073] (3) Use an eight-pipette to draw bacterial culture from the plate and add it to a 96-well plate, 50 μL per well, up to column 11. Add MHB blank broth to column 12 as a negative control.
[0074] (4) Add FFC 10 μL to the first column of the 96-well plate to make the final concentration 512 μg / mL, and after mixing by blowing, take 50 μL of the mixture to the next column for dilution by half.
[0075] (5) Seal the 96-well plate with a sealing film and place it in a 37°C incubator for 20 h.
[0076] (6) Observe the results and record the minimum inhibitory concentration. The results are shown in Table 1. Figure 7
[0077] 2 Checkerboard drug susceptibility test
[0078] (1) After 19K-1068, 19K-826, 19K-69, 20K-106, 19K-202, 20K-196, 19K-1130, 20K-449, 19K-979, 19K-845, 19K-271, 19K-847, 768 and 700603 were cultured on MH agar plates at 37°C for 10 h, single colonies were picked and cultured in 3 mL of fresh MH broth at 37°C for 5 h.
[0079] (2) Take 100 μL of the bacterial solution and mix it with 900 μL of fresh MH broth. Then take 20 μL of the bacterial solution and dilute it 1,000-fold in 20 mL of MHB broth in a 90 mm plate, and mix by blowing with an eight-way pipettor.
[0080] (3) Use an eight-way pipettor to extract the bacterial solution from the plate and add it to the 96-well plate, 50 μL per well, for a total of 11 columns.
[0081] (4) Add FFC 10 μL to the first column of the 96-well plate to make the final concentration 512 μg / mL, and after mixing by blowing, take 50 μL of the mixture to the next column for dilution by half. Dilute to the 11th column. Set the concentration of compound T to 7 gradients, and use a 2 mL EP tube to dilute by half to obtain 7 concentrations from large to small.
[0082] (5) Add the corresponding T concentrations from high to low to each layer of the 96-well plate from top to bottom, a total of 7 rows and 11 columns. At this time, the 12th column of the 96-well plate is the T single-drug positive control, and the 8th row is the polymyxin B positive control.
[0083] (6) Seal the 96-well plate with a sealing film and place it in a 37°C incubator for 24 h.
[0084] (7) Observe the results and record the MIC.
[0085] (8) Calculate the inhibitory concentration FIC index: FIC = MIC(A combination) / MIC(antibiotic alone) + MIC(B combination) / MIC(compound alone).
[0086] (9)Result determination: FIC <0.5, synergistic effect; when FIC is between 0.5-1, additive effect; when FIC is greater than 1 and less than 2, irrelevant effect; when FIC > 2, antagonistic effect.
[0087] The results are as follows Figure 8 , Compound T has synergistic effect when combined with polymyxin B, and the FIC of 19K-1068, 19K-826, 19K-69, 20K-106, 19K-202, 20K-196, 19K-1130, 20K-449, 19K-979, 19K-845, 19K-271, 19K-847, 768 and 700603 are all less than 0.5, which has synergistic effect, and the MIC of polymyxin B is reduced by 4-16 times; when combined with gentamicin, the FIC of 19K-202, 19K-1130, 19K-979, 19K-845, 19K-271, 19K-847 are 0.5, 0.5, 0.5, 0.5, 0.25, 0.5 respectively, which has additive effect; when combined with amikacin, ciprofloxacin, doxycycline, enrofloxacin, ceftiofur and tigecycline, most of them have antagonistic effect. The results show that T can achieve better effect when combined with polymyxin B.
[0088] 3Time-kill curve
[0089] (1) Inoculate 19K-1068, 19K-979 on LB agar plates and incubate at 37°C for 8h;
[0090] (2) Pick single colonies into LB broth and incubate to the logarithmic growth phase;
[0091] (3) Set 6 groups, respectively, polymyxin B concentration is 1 / 4 MIC, T concentration is 1 / 4 MIC and 1 / 8 MIC, combination of polymyxin B and 1 / 4 MIC T, combination of polymyxin B and 1 / 8 MIC T, blank control group without adding antibiotics. Add 30mL MH broth to each group, add bacteria solution diluted to 1×10 7 CFU / mL in advance, and add the set antibiotic concentration, and place each group in a 37°C shaking incubator. Take 1mL bacterial solution at 0, 4, 8, 12, 24h to measure OD 540 , dilute 3 gradients according to OD, take 100μL bacterial solution of each gradient and spread on plates (set 3 parallel), and incubate in a 37°C constant temperature incubator for 10h. Observe the number of colonies and count, and draw the time-kill curve.
[0092] (4) Result determination: compared with each drug alone, the average bacterial growth of the combination of the two drugs is reduced by ≥2Log 10CFU / mL was determined to be a synergistic effect; bacterial growth decreased by an average of 1–2 Log. 10 CFU / mL was considered an additive effect; the average decrease in bacterial growth was ≤1 Log. 10 CFU / mL was considered an irrelevant effect; while the average increase in bacterial growth was ≥2 Log. 10 If the concentration of CFU / mL is high, it is considered an antagonistic effect.
[0093] like Figure 9 and Figure 10 As shown, for 19K-1068, when polymyxin B (0.00390625 μg / mL) was used in combination with a low concentration of T (0.03125 mg / mL), the bactericidal effect was not significantly enhanced; however, when polymyxin B (0.00390625 μg / mL) was used in combination with a high concentration of T (0.625 mg / mL), the bactericidal effect was significantly improved, and the number of strains decreased by 4.43 log within 24 hours, showing a clear synergistic bactericidal effect. For strain 19K-979, the combined use of polymyxin B (0.0153125 μg / mL) and low concentration T (0.03125 mg / mL) showed a certain synergistic effect, with the number of strains decreasing by 2.6 log within 24 hours. When the concentration of polymyxin B was reduced to 0.00390625 μg / mL and combined with high concentration T (0.625 mg / mL), the synergistic bactericidal effect was further enhanced, with the number of strains decreasing by 5.03 log within 24 hours, demonstrating a significant bactericidal effect.
[0094] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings and specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.
Claims
1. A method for determining the binding site of an LpxC enzyme inhibitor in Klebsiella pneumoniae, characterized in that, Comprise the following steps: 1) select different homology levels of Klebsiella pneumoniae strains, and use SWISS MODE website to model homology and optimize GalaxyRefine to obtain LpxC enzyme protein model; 2) verify the binding sites obtained from software and literature, evaluate the advantages and disadvantages of the binding pocket according to the obtained 2D structure diagram, and obtain the LpxC enzyme protein binding pocket.
2. The method for determining the binding site of the Klebsiella pneumoniae LpxC enzyme inhibitor according to claim 1, characterized in that, In step 1), the Klebsiella pneumoniae strains with different homology levels are selected from the strains with different homology levels, including human and animal sources.
3. The method for determining the binding site of the Klebsiella pneumoniae LpxC enzyme inhibitor according to claim 1, characterized in that, In step 2), the determination of the LpxC enzyme protein binding pocket is based on the comparison of mature hydroxyl amide compounds to verify the advantages and disadvantages of these pockets according to the literature and software generated binding sites.
4. The method for determining the binding site of the Klebsiella pneumoniae LpxC enzyme inhibitor according to claim 1, characterized in that, In step 2), four LpxC enzyme protein binding pockets are determined, and the key amino acids are as follows: Binding pocket (1): L143, A160, P161, P192, P194, L239, A242, L262, H265; Binding pocket (2): L18, C63, E78, H79, T191, F192, M195, I198, F212, V217, H238, K239, D242, H265; Binding pocket (3): E78, H79, T191, H238, D242, D246, H265; Binding pocket (4): L18, H19, M61, L62, C63, T76, G78, H79, I103, G106, A190, T191, P192, G193, P194, M195, A196, A197, I198, G199, T200, L201, G202, L206, C207, L208, G209, G210, S211, P212, C214, A215, I216, V217, V218.
5. A method of screening for inhibitors of a Klebsiella pneumoniae LpxC enzyme, comprising, contacting a test compound with a LpxC enzyme, and determining whether the test compound inhibits the LpxC enzyme. Comprise the following steps: 1) select different homology levels of Klebsiella pneumoniae strains, and use SWISS MODE website to model homology and optimize GalaxyRefine to obtain LpxC enzyme protein model; 2) verify the binding sites obtained from software and literature, evaluate the advantages and disadvantages of the binding pocket according to the obtained 2D structure diagram, and obtain the LpxC enzyme protein binding pocket; 3) For the determined LpxC enzyme protein model and LpxC enzyme protein binding pocket, select the classic ZINC15 natural compound database for high-throughput screening to obtain the primary screening small molecules; 4) screen the primary screening small molecules for ADMET and drug-like properties to ultimately obtain the target inhibitor.
6. The method of screening for inhibitors of Klebsiella pneumoniae LpxC enzyme according to claim 5, wherein, In step 3), the screening standard of ZINC15 natural compound database is Affinity≤-7kcal / mol.
7. The method for screening Klebsiella pneumoniae LpxC enzyme inhibitors according to claim 5, characterized in that, In step 4), The ADMET screening criteria are: aqueous solubility of 4 (-2.0 < log(Sw) < 0.0); blood brain barrier penetration of 1 (0 < logBB < 0.7); intestinal absorption of 1; no hepatotoxicity; no binding to plasma proteins; no inhibition of CYP2D6; no skin irritation; The drug-like screening criteria are based on the Lipinski's Rule of Five, which are: no more than 5 hydrogen bond donors; no more than 10 hydrogen bond acceptors; no more than 5 logP; molecular weight below 500; no more than 10 rotatable bonds.
8. Use of a LpxC enzyme inhibitor obtained by the screening method of any one of claims 5 to 7 in the manufacture of a medicament for inhibiting Klebsiella pneumoniae.
9. Use of 5-methyl-3-hexen-2-one in the manufacture of a medicament for inhibiting Klebsiella pneumoniae.
10. Use of 5-methyl-3-hexen-2-one in combination with an antibacterial agent in the manufacture of a medicament for inhibiting Klebsiella pneumoniae; preferably, the antibacterial agent is polymyxin B.