Use of chembridge 53717615 in the preparation of an acinetobacter baumannii adeabc efflux pump inhibitor

Abstract

The application discloses application of Chembridge 53717615 in preparation of an Acinetobacter baumannii AdeABC efflux pump inhibitor, and a molecular formula of the Chembridge 53717615 is C 19 H 25 N5O2 The application discloses a method for discovering, based on computer-aided drug design, a compound Chembridge 53717615 which can inhibit the uptake and efflux of antibiotics by an Acinetobacter baumannii efflux pump and also has an inhibiting effect on the formation of a biological membrane, can greatly reduce antibiotic resistance, has important significance for treating clinical drug-resistant Acinetobacter baumannii infection, and has a great application and development prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibacterial efflux pump inhibitors, and particularly relates to application of Chembridge 53717615 in preparation of an Acinetobacter baumannii AdeABC efflux pump inhibitor. BACKGROUND

[0002] Acinetobacter baumannii (AB) is the most common gram-negative bacillus in Acinetobacter, which widely exists in water, soil and hospital environment, is an important pathogenic bacterium causing nosocomial infection, and can cause infection of multiple tissues and organs of the body, such as urinary tract infection, respiratory-related pneumonia, wound infection, soft tissue infection, etc. In recent years, due to the irrational use of antibiotics, the detection rate of drug-resistant strains of Acinetobacter baumannii has increased dramatically worldwide, and the drug resistance of multidrug-resistant Acinetobacter baumannii (MDR-AB) has become more and more serious.

[0003] Due to the widespread abuse of antibacterial drugs, Acinetobacter baumannii can develop resistance to almost all antibiotics, which brings great challenges to clinical treatment. The types of drug resistance mechanisms of Acinetobacter baumannii are complex and diverse, mainly including intrinsic resistance and acquired resistance, including production of β-lactamase, overexpression of efflux pump, change of drug action target, decrease of protein pore channel expression, formation of biofilm, etc.

[0004] (1) Production of β-lactamase

[0005] β-lactamase is a kind of protein that can hydrolyze the β-lactam ring of this kind of antibiotic, making it break and lose antibacterial activity, which is one of the important drug resistance mechanisms of Acinetobacter baumannii.

[0006] According to Ambler classification, it is mainly divided into A, B, C, D, i.e. A class of super broad-spectrum β-lactamase (ESBLs), B class of metalloenzyme, C class of cephalosporinase (AmpC), D class of OXA type enzyme. The main ones related to the production of drug resistance of Acinetobacter baumannii include A, B and D classes of enzymes, and C class of enzymes is rarely produced.

[0007] (2) Overexpression of efflux pump

[0008] Overexpression of efflux pumps is one of the main mechanisms of multidrug resistance in Acinetobacter baumannii, which can reduce the content of drugs in the bacterial body, leading to the failure of antibacterial drugs to exert antibacterial effect and cause drug resistance. There are five families of efflux pumps known to be associated with increased bacterial resistance: ATP binding cassette superfamily (ABC), Resistance nodulation division family (RND), small multidrug resistance family (SMR), Multidrug and Toxic compound Extrusion family (MATE), and Major facilitator superfamily (MFS).

[0009] Research reports that the Resistance nodulation division family is the most important efflux system causing Acinetobacter multi-drug resistance, mainly including AdeABC, AdeFGH and AdeIJK efflux pumps, among which AdeABC efflux pump is the earliest efflux system studied in clinical research and is the most important factor causing drug resistance.

[0010] (3) Change of drug action target

[0011] After the modification or gene mutation of the drug action target, the affinity between the drug and the target will decrease, leading to the generation of bacterial drug resistance. Typical penicillin-binding proteins (PBPs) are the target of carbapenem antibiotics, which mainly inhibit the synthesis of cell wall mucin by inhibiting the synthesis of cell wall mucin, thereby hindering the synthesis of cell wall mucin, causing bacterial cell wall defects, and causing bacterial cytoplasmic osmotic pressure changes and cell lysis to kill bacteria. PBPs reduce the binding ability of carbapenem antibiotics by changing the drug action site, and the antibacterial ability of Acinetobacter baumannii is enhanced, leading to the generation of CRAB.

[0012] (4) Loss or change of outer membrane protein

[0013] Outer membrane porin is the channel for bacteria to exchange various substances, and outer membrane protein is involved in the transport of various antibacterial drugs. The deletion or alteration of outer membrane protein is one of the important mechanisms of multidrug-resistant Acinetobacter baumannii. When the outer membrane protein gene changes, the expression of protein pore channel decreases, the pore through which the drug passes will be blocked or disappear, which will reduce the permeability of the bacterial outer membrane, hinder the entry of drugs into the bacterial body, and lead to bacterial drug resistance. For example, outer membrane protein A (Omp A) is the main component of the outer membrane protein of gram-negative bacteria, which plays an important role in the drug resistance of Acinetobacter baumannii. It can change the permeability of the outer membrane and invade the target cells to cause apoptosis by binding to specific components on the cell surface.

[0014] (5) Formation of biofilm

[0015] Biofilm is a physical barrier formed by bacterial cell groups wrapped in extracellular polymers such as polysaccharides, proteins, nucleic acids, and lipids. It can enhance the resistance of bacteria to antibacterial drugs. The strong adhesion of Acinetobacter baumannii makes it easy to form biofilm on hospital environment and various medical devices, which can produce drug resistance. The main mechanism is that the high-density bacteria in the biofilm can prevent antibacterial drugs from entering the cell matrix to some extent, making it difficult for antibacterial drugs to act, and the bacteria can produce strong drug resistance and immune escape ability. In addition, the lack of nutrients and air in the biofilm makes the bacteria grow slowly and weakens their sensitivity to antibiotics.

[0016] Acinetobacter baumannii efflux pump-AdeABC

[0017] Efflux pump (EP) is a protein transport system that expels intracellular drugs or toxic substances out of the cell, which requires proton exchange or ATP hydrolysis to provide energy. Among the five major efflux systems, the RND family efflux system plays an important role in the formation of multidrug-resistant Acinetobacter baumannii, mainly involving AdeABC, AdeFGH, and AdeIJK. The efflux system of this family is mainly composed of inner membrane transport protein (IMP), periplasmic fusion protein (MFP), and outer membrane efflux protein (OEP). IMP and OEP form a homotrimer structure, and MFP forms a hexamer structure. IMP utilizes the proton concentration gradient to provide energy and expels the substrate through the proton / substrate antiporter mechanism. In the RND family, efflux pump AdeABC is the most important member, which was first discovered in Acinetobacter baumannii and has been studied the most in recent years. It can expel various antibacterial drugs such as β-lactams (including carbapenems), aminoglycosides, fluoroquinolones, macrolides, and tetracyclines. Overexpression of the AdeB efflux gene may even lead to resistance to carbapenem antibiotics, which are the first choice of clinical drugs.

[0018] AdeABC is a triad of membrane fusion protein (AdeA), multidrug transporter protein (AdeB) and outer membrane channel protein (AdeC). In the process of efflux, AdeA mainly coordinates the structure of outer membrane protein and makes the inner and outer membrane of bacterial cell as close as possible; AdeB is the main functional element of efflux pump, which can recognize and uptake drugs and actively transport them to the cell membrane through AdeC. The AdeABC operon is located in the chromosomal DNA and maintains low expression in Acinetobacter baumannii. Its expression is strictly regulated by the regulatory gene (AdeRS). AdeRS is a classic two-component regulatory system, AdeS is a signal recognition protein, and AdeR is a DNA binding protein. Both are encoded by the AdeRS operon upstream of the AdeABC operon and transcribed in opposite directions, and they jointly regulate the expression of the AdeABC gene. Overexpression of AdeABC efflux pump can increase the efflux of antibiotics and reduce the accumulation of intracellular antibiotics, thereby increasing the drug resistance and multidrug resistance of Acinetobacter baumannii. Studies have shown that the drug resistance of Acinetobacter baumannii is related to the high expression of AdeB gene, and AdeABC can mediate carbapenem antibiotic resistance together with outer membrane protein. In 2018, Zhang Yanpeng et al. confirmed that the overexpression of Acinetobacter baumannii efflux pump gene AdeB is closely related to the drug resistance mechanism of carbapenem antibiotics by real-time fluorescent quantitative detection of efflux pump gene expression. Therefore, in view of the current serious problem of bacterial drug resistance, we can develop new drugs based on the target AdeB to inhibit the overexpression of efflux pump and solve the problems of drug resistance and multidrug resistance of Acinetobacter baumannii.

[0019] In the past decade, active efflux is a major cause of antibiotic resistance and one of the most important trends in anti-infective therapy. The substrate versatility of efflux systems makes this phenomenon almost affect all kinds of antibiotics. Usually, the overexpression of efflux genes leads to increased drug efflux, decreased intracellular drug concentration, and reduced clinical efficacy, thereby mediating the emergence of multidrug resistance phenotype and being an important mechanism of multidrug resistance. Efflux pump inhibitors (EPIs) can inhibit the efflux of drugs by resistant bacteria, restore their sensitivity to drugs and improve the treatment effect of resistant bacteria.

[0020] At present, many compounds have been found to inhibit the efflux of efflux pump. According to the different energy supply modes of active efflux system, they can be divided into ATP hydrolysis energy driven type, transmembrane proton gradient energy driven type and other natural compound inhibitors.

[0021] 1) ATP hydrolysis energy driven type

[0022] Reserpine (RES), a small molecule alkaloid with hypotensive activity, is the first compound found to inhibit bacterial efflux. Neyfakh et al. reported that reserpine could inhibit bacterial efflux, improve the susceptibility of Staphylococcus aureus to norfloxacin, ciprofloxacin and other efflux pump substrates, and inhibit both intrinsic and antibiotic-induced efflux pumps, while reducing the emergence of resistant strains. This study has injected new energy into solving the problem of drug-resistant bacterial infections and given a new direction for the discovery of subsequent efflux pump inhibitors.

[0023] Similarly, Verapami (VER), used to treat hypertension and other diseases, is a voltage-gated type I calcium channel blocker and a specific inhibitor of the efflux transporter P-glycoprotein and cytochrome P450 3A4 enzyme (CYP3A4). It has been reported that it can reduce the minimum inhibitory concentration of various anti-mycobacterial drugs by blocking the efflux pump of Mycobacterium tuberculosis, and, like some membrane-active agents, verapamil disrupts membrane function and induces membrane stress, thereby producing a synergistic effect with anti-tubercle bacillus drugs, but its efflux pump inhibition mechanism has not been fully elucidated.

[0024] (2) Transmembrane proton gradient-driven

[0025] Phenylalanyl arginyl β-naphthylamide (PAβN) is a broad-spectrum inhibitor that is effective against various Gram-negative bacterial resistance-nodulation-division (RND) pumps and restores the antibiotic sensitivity of strains. In particular, it can effectively reduce the efflux of fluoroquinolones by the three RND pumps, MexAB-OprM, MexCD-OprJ and MexEF-OprN, of Pseudomonas aeruginosa, and also has an inhibitory effect on the AcrAB-TolC pump of Escherichia coli.

[0026] Opperman et al. reported that its inhibitory effect may be the result of competition with drugs for binding sites during transport. Misra et al. ultimately concluded that the main mechanism of action of PAβN is to inhibit efflux pump activity. PAβN has good inhibitory effect on various efflux systems. However, the cationic group in its structure can cause long-term accumulation of the drug in tissues, leading to severe kidney toxicity. Although attempts have been made to improve its drug properties by modifying its structure, no substantial progress has been made. In addition, carbonyl cyanide 3-chloropenylhydrazone (CCCP), which also has unique biological activity in reversing polymyxin resistance, is also in a similar situation.

[0027] (3) Other natural compound types

[0028] Tetrandrine (TET), a bisbenzylisoquinoline alkaloid, is extracted from the traditional Chinese herb Stephania tetrandra S. Moore and has been used to treat cancer, inflammation, asthma, and hypertension. Similar to verapamil, tetrandrine is a typical natural L-type calcium channel and P-glycoprotein inhibitor. Zhang et al. found that tetrandrine combined with isoniazid (INH) and ethambutol (EMB) can reduce the minimum inhibitory concentration of multidrug-resistant Mycobacterium and help reduce drug dosage and side effects. 2+

[0029] In addition, Berberine (BBR) is an amphipathic isoquinoline alkaloid widely present in plants of the genus Berberis, which has antibacterial, antioxidant, anti-inflammatory, anti-cholesterol, and anti-diabetic pharmacological effects, and is also a recognized substrate of efflux pumps. When combined with antibiotics, the intracellular concentration of berberine increases, and due to the competitive inhibition of efflux, the effect of antibiotics is also enhanced, ultimately resulting in enhanced overall antibacterial effect. Given the limited solubility of berberine, poor gastrointestinal absorption, and low plasma levels due to severe first-pass metabolism and P-glycoprotein-mediated efflux, it has been puzzling how oral berberine can exert such a wide range of pharmacological effects in the body.

[0030] With the increasing infection of Acinetobacter baumannii and multidrug resistance, efflux pumps have become a key factor in combating drug resistance. Most of the reported efflux pump inhibitors are selected from existing drugs, although these inhibitors can improve the sensitivity of antibacterial drugs, but their multiple adverse reactions, low biological activity and other shortcomings limit their use as antibacterial drug enhancers for anti-infection treatment. The understanding of efflux pump inhibitors provides a new idea and lays a certain theoretical foundation for solving the problem of drug-resistant bacterial infection, and efflux pumps are not only related to the efflux of various antibiotic drugs, but also involved in physiological activities such as strain movement and toxicity production, and the development of their inhibitors is expected to become the key to solving the problem of drug resistance and multidrug resistance. Therefore, more and more researchers have paid attention to the development of new efflux pump inhibitors based on potential targets of efflux systems. SUMMARY

[0031] To solve the above technical problems, the purpose of the present application is to provide an application of Chembridge 53717615 in preparing an Acinetobacter baumannii AdeABC efflux pump inhibitor, which has a molecular formula of:

[0032] The structural formula is: .

[0033] ​The present application is based on the method of computer-aided drug design to find the compound Chembridge 53717615 which can inhibit the uptake and efflux of antibiotics by Acinetobacter baumannii efflux pump, and also has inhibitory effect on biofilm formation, can greatly reduce antibiotic resistance, has important significance for the treatment of clinically drug-resistant Acinetobacter baumannii infection, and has great application and development prospects. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The compound docking conformation diagram.

[0035] Figure 2 The simulation results of the complex of the control compound CCCP and the protein AdeB.

[0036] Figure 3 The simulation results of the complex of the compound Chembridge 53717615 and the protein AdeB.

[0037] Figure 4 The growth curves of Acinetobacter baumannii and multi-drug resistant Acinetobacter baumannii.

[0038] Figure 5 The drug sensitivity experiment.

[0039] Figure 6 The in vitro toxicity of the compound on HSF cells.

[0040] Figure 7 The ADME\T prediction results of 12 compounds.

[0041] Figure 8 The RMSF, SASA and Rg results of 12 complex systems in the process of MD simulation. DETAILED DESCRIPTION

[0042] The present application will be further described below in combination with examples.

[0043] The present application selects the main element AdeB of Acinetobacter baumannii AdeABC efflux pump as the target protein, and its crystal structure (PDB ID: 7KGG, resolution: 1.9 Å) is obtained from the PDB protein database (http: / / www.rcsb.org / pdb). The compound of the present application is selected from ChemDiv and Chembridge databases, and the small molecule compound Chembridge 53717615 is mainly from the database: Chembridge.

[0044] The molecular formula is:

[0045] The structural formula is: .

[0046] Example 1

[0047] Docking of Chembridge 53717615 compound with receptor protein molecule

[0048] The software used SYBYL-X 2.0 (Shanghai YUAN Capital Information Technology Co., Ltd.), Discover Studio (Accelrys Software Inc.), etc. to perform virtual screening, molecular docking and molecular simulation studies.

[0049] 1) Pretreatment of receptor protein

[0050] Based on the Surflex-Dock module in SYBYL-X2.1.1 software, the AdeB crystal structure obtained from the PDB protein database was pretreated. First, the secondary structure of the receptor protein was observed, and the ligand molecule in the complex was extracted into the Prepare Protein Structures protein preparation module; then the two modules of Remove Substructure and Analyze Selected Structure were used to complete the removal of water molecules in the crystal protein, repair of amino acid residues, etc. Finally, the pretreated receptor protein was saved in pdb format.

[0051] 2) Pretreatment of ligand

[0052] The SYBYL-X2.1.1 software was used to load the force field, energy minimization, etc. on 12 compounds including Chembridge 53717615 (see Table 1) and save the ligand file.

[0053] (3) Molecular docking

[0054] The pretreated receptor protein was loaded through the Surflex-Dock module, and the docking configuration file was created based on the extracted co-crystal ligand. Mainly using Surflex-Dock Screen (SFXC) and Surflex-Dock GeomX (SFXC) 2 modes in Docking Mode to complete. First, the rigid docking module (Surflex-dock) was used to preliminarily screen the molecules, and 12 compounds in Table 1 were obtained, then the drug-like properties of the compounds obtained by preliminary screening were evaluated, and the Surflex-Dock GeomX module was used to dock the compounds with drug-like properties. High-precision docking was performed on the compound. Then the compound was subjected to molecular dynamics simulation and binding free energy evaluation.

[0055] 4) Drug-like property evaluation

[0056] ​The compound Chembridge 53717615 obtained by the initial screening of the molecules was subjected to drug-like property evaluation using the software Discover Studio. The compound was loaded into the molecular window interface, and the FilterLigands tool in the Small Molecules module was used to filter the small molecule compound. This method is mainly based on the Lipinski and Veber rules to evaluate the drugability, and needs to meet the requirements of compound molecular weight ≤500 Da, octanol-water partition coefficient Log P ≤5, number of hydrogen bond donors ≤5, number of hydrogen bond acceptors ≤10, and number of rotatable bonds ≤10, etc.

[0057] 5) Molecular dynamics simulation

[0058] Before MD simulation, we obtained the relevant data of the spatial arrangement of the protein in the lipid bilayer from the OPM website (https: / / opm.phar.umich.edu). And we used the CHARMM-GUI web server to embed the complete AdeB protein structure into the inner membrane of Acinetobacter baumannii, with a size of about 111 × 111 × 111 nm (www.charmm-gui.org). We chose a simplified version of the Acinetobacter baumannii inner membrane, which is composed of 80% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 20% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG).

[0059] PMEMD.mpi and PMEMD.cuda modules were used. First, the receptor protein AdeB and the compound were loaded with ff14SB force field and Amber force field (GAFF), respectively, and the initial conformation of the complex system was inserted into a water tank with a TIP3P solvation model, with a cubic box carrier of 112 × 112 × 112 nm, and sodium ions were added under periodic boundary conditions to neutralize the charge of the entire system. Then, in order to avoid unfavorable collisions between molecules in the complex, the conjugate gradient algorithm and the steepest descent method were used for 5000 cycles of minimization processing, and the temperature and pressure of the complex system were stabilized at 300 k and 1 atm (time step of 2 fs) respectively through NVT and NPT simulation. Finally, 100 ns of dynamics simulation was performed for each complex system, and the motion trajectory of the simulation system was saved every 100 ps.

[0060] 6) Binding free energy calculation

[0061] Based on the trajectory file of the dynamics simulation, the MM / GBSA method was used to calculate the binding free energy between the candidate compound and the AdeB target, and to decompose the energy contribution of each amino acid residue, with the following formula:

[0062]

[0063] wherein is the binding energy, is the sum of the molecular mechanical energies in vacuum, whose energy contributions include electrostatic energy, van der Waals energy and internal energy; is the energy of solvation, including polar solvation energy ( ) and nonpolar solvation energy ( ); is the change in the conformational entropy of the ligand, is the absolute temperature, is the entropy of the molecule.

[0064] 7) ADME\T property prediction

[0065] The ADME\T property of the compound was predicted theoretically by using Discover Studio software, mainly including Absorption, Distribution, Metabolism, Excretion indicators, etc. In the ADME\T Descriptors module, we selected the ADME\T descriptors Aqueous solubility, Cytochrome P450 inhibition, BBB, HIA, Hepatotoxicity and PPB to predict the pharmacokinetic properties of the compound.

[0066] From the docking score, the results of ChemDiv L676-2179 and ChemDiv L676-1461 were obviously better than the control drug CCCP (Total Score = 3.802), Chembridge L676-2179 had a high docking score (Total Score = 7.214), and ChemDiv L676-1461 was the second, indicating that the selected compounds may also have better biological activity.

[0067] Table 1 Chemical structure and docking score of 12 compounds

[0068]

[0069] Figure 1 shows the docking conformation of 12 compounds, 12 compounds are located in the target protein Chembridge 53717615 can also form a good fit with the receptor protein in the spatial structure. In addition, there are important amino acid residues Met570, Met656, Val658, Phe612, Glu710, Trp708 and Met706 present at the binding site, which play a key role in the mutual binding between the compound and the target protein.

[0070] 8) Drug-likeness prediction results

[0071] Based on the drug-likeness prediction of the compound by Discover Studio, the evaluation results of the compound are shown in Table 2. The molecular weight, octanol-water partition coefficient, number of hydrogen bond donors, number of hydrogen bond acceptors and number of rotatable bonds of the compound are basically within the reasonable range. Therefore, subsequent molecular dynamics simulation is carried out to explore the binding mechanism of the target and the candidate compound and other researches.

[0072] Table 2 Drug properties of the compound

[0073]

[0074] 9) Results of molecular dynamics simulation

[0075] The complex of the protein and the candidate compound is subjected to 100 ns molecular dynamics simulation by using AMBER software, so as to obtain the stable binding conformation of the compound and the protein, and to further study the interaction mode of the compound and the protein and the energy contribution of the key amino acid residues. The simulation results of the complex of the control compound CCCP and the protein AdeB are shown in Figure 2 . Fig. 2a is the root mean square deviation (RMSD) of the AdeB / CCCP complex dynamics simulation. At the beginning of the simulation, CCCP fluctuates up and down around ~0.5 and ~1.5 Å, and is balanced around ~1.0 Å after 85 ns. The AdeB protein has too many random coil regions in its structure, resulting in a high RMSD value. From the initial fluctuation position of ~5.0 Å, it slowly rises to ~6.5 Å after 30 ns, and then remains stable at this value. It is shown that the complex system has reached a stable state. Figure 2Figure 2b shows the binding free energy contribution of key amino acid residues in the AdeB / CCCP complex. Ten residues located at the binding site—Val707, Val658, Trp708, Ser572, Pro660, Phe612, Met706, Met656, Met570, and Ile821—contribute significant energy, with Val658 and Pro660 contributing -6.941 kcal / mol and -4.190 kcal / mol, respectively. Figure 2c shows the hydrogen bond analysis results of the AdeB / CCCP complex. During the entire dynamic simulation, the AdeB / CCCP complex formed 1-2 hydrogen bonds. Figure 2 The ef parameter represents the interaction mode of the lowest energy conformation of the AdeB / CCCP complex. The compound CCCP forms one hydrogen bond and one hydrophobic interaction with the AdeB protein residues Val658 (5.3 Å, 2.7 Å), respectively; and three hydrophobic interactions with residues Leu659 (4.2 Å), Pro660 (4.2 Å), and Pro661 (5.1 Å). Furthermore, it forms a halogen bond with Met561 (3.4 Å) and a Pi-sulfur bond with Met706 (4.2 Å).

[0076] The complex system of compound Chembridge 53717615 and AdeB was relatively stable during 100 ns MD simulations, such as... Figure 3 As shown in Figure 3a, the RMSD results of the system are as follows: the complex enters a stable state after 20 ns, Chembridge53717615 fluctuates smoothly around ~2.4 Å, while AdeB slowly rises between ~5.0 Å and ~5.5 Å in the first 20 ns, and then stabilizes and fluctuates around 5.5 Å. Figure 3 b shows the binding free energy contribution of key amino acid residues in the AdeB / Chembridge 53717615 complex. Compared with the control CCCP, it has most of the same amino acid residues, with the interaction energies of Ser572 and Met656 being -4.624 kcal / mol and -5.244 kcal / mol, respectively. Additionally, two residues, Leu712 and Gln574, show significant energy contributions at the binding site, with values ​​of -6.342 kcal / mol and -3.980 kcal / mol, respectively. The number of hydrogen bonds during the simulation is shown below. Figure 3 As shown in c, the overall value remains between 1 and 3. Figure 3e is the interaction mode of the lowest energy conformation of the AdeB / Chembridge 53717615 complex, the compound Chembridge 53717615 forms 5 hydrogen bonds with AdeB protein residues Gln574 (3.0 A), Gln805 (3.0 A), Ser572 (3.0 A), Ser613 (3.0 A), Met656 (3.4 A) respectively, and 5 hydrophobic interactions with Val619 (4.1 A), Leu712 (4.3 A, 4.8 A), Met656 (4.3 A, 5.4 A), in addition, it also forms 1 Pi-Sulfur bond with Met656 (3.1 A).

[0077] 10) Results of RMSF, RG and SASA

[0078] To further evaluate the stability of the 12 complex systems, we calculated the root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) of the molecular dynamics simulation trajectories, the results are shown in Figure 8 Figure 8 is the RMSF plot of the AdeB receptor protein of the 12 complex systems, which intuitively shows the flexibility of the backbone structure and the local motion characteristics of the structure. Obviously, during the simulation, because ChemDiv C200-6341 and ChemDiv 3379-1103 cannot bind well with the receptor protein, it causes the protein to vibrate greatly, resulting in large fluctuations in the amino acid residue RMSF of the complex AdeB / ChemDiv C200-6341 and AdeB / ChemDiv 3379-1103. While the RMSF values of most residues of the other 10 complexes are less than 4 A, and the receptor proteins have similar fluctuation trajectories, indicating that the protein AdeB bound with these compounds has good dynamic stability. The radius of gyration characterizes the tightness of the particle binding and can reflect the extensibility of the protein in the spatial distribution, when the tightness of the particle binding is larger, the corresponding Rg value is smaller, and the results are shown in Figure 8 c. The Rg values of the backbone atoms of the 12 systems are basically stabilized at about ~36 A to ~38 A, respectively, which means that the extensibility of the protein is good during the simulation. Figure 8 b is the change of the solvent accessible surface area of the complex with time, the SASA values of the hydrophobic amino acid residues of the 12 complexes are stable around ~70000 A 2 , indicating that the number of hydrophobic amino acids hidden inside the protein is basically unchanged, and the structure of the protein is stable during the simulation time.

[0079] 11) Results of binding free energy calculation

[0080] ​Since drug action is also closely related to binding affinity, and binding free energy can be used to represent the interaction ability between ligand and acceptor, the binding free energy of 12 complexes was calculated using the MM / GBSA method, mainly including electrostatic interactions, van der Waals forces, and other forces generated between the complexes. The results are shown in Table 3.

[0081] The binding energies of the 12 candidate inhibitors, in descending order, are as follows: ChemDiv L676-2179 > ChemDiv L676-1461 > Chembridge 53717615 > ChemDiv C200-6341 > Chembridge 93546223 > ChemDiv T482-1954 > ChemDiv 6809-0355 > ChemDiv D072-0756 > ChemDiv 2641-1576 > ChemDiv F432-0587 > ChemDiv D621-0003 > ChemDiv 3379-1103. Compared to the binding energy of CCCP (ΔGBind = -28.94 Kcal / mol), compounds ChemDiv L676-2179, ChemDiv L676-1461, and Chembridge 53717615 exhibit significantly superior binding energies, at -42.083 Kcal / mol, -40.978 Kcal / mol, and -36.568 Kcal / mol, respectively. This suggests that they bind more readily to receptor proteins and may possess superior potential biological activity.

[0082] Table 3 shows the results calculated using the combined free energy (Kcal / mol).

[0083] Table3 Results of bind free energy calculation (Kcal / mol).

[0084]

[0085] 12) ADME\T prediction results

[0086] We performed ADME\T prediction analysis on 12 candidate compounds, and the results are as follows: Figure 7The compounds are shown. Except for compound Chembridge 93546223, the rest of the compounds are within the defined ellipse range, which indicates that they meet the requirements of the five principles of drug-like, i.e. 11 have good ADME\T characteristics, and Chembridge 53717615 meets the characteristics of compound drug. Therefore, we will synthesize this series of compounds as potential efflux inhibitors, and verify their potential biological activity through in vitro activity experiments.

[0087] Example 2

[0088] Biological activity evaluation

[0089] 1) Materials

[0090] (1) Experimental strain: Wild-type Acinetobacter baumnnii (Acinetobacter baumnnii GDMCC-11336) was purchased from the Guangdong Provincial Microbial Culture Collection Center, and drug-resistant Acinetobacter baumnnii was provided by the Army Medical University.

[0091] (2) Cell line: HSF cells (human normal epidermal fibroblasts) were stored in the laboratory (Chongqing University Cancer Hospital).

[0092] 2) Solution preparation

[0093] (1) Preparation of solid culture medium: The required amount was prepared in the ratio of NA solid powder: distilled water = 33 g: 1000 mL. After autoclaving, pour into the plate when cooled to about 40°C. Solidify for use.

[0094] (2) Preparation of liquid culture medium: The required amount was prepared in the ratio of MH broth solid powder: distilled water = 24 g: 1000 mL. Use after autoclaving and cooling.

[0095] (3) Preparation of phosphate buffer: Take 1 bag of phosphate buffer powder, completely dissolve in 1 L of secondary water, autoclave at 121°C for 20 min, and then sub-pack for use.

[0096] (4) DMEM complete medium: DMEM medium (90 mL) + 10% fetal bovine serum (10 mL) + 0.1% double antibody (1 mL).

[0097] (5) Preparation of compounds: Dissolve an appropriate amount of CCCP, candidate inhibitors, imipenem, cephradine, levofloxacin, amikacin, tetracycline, azithromycin, and gentamicin in dimethyl sulfoxide (cell grade) or sterilized water to prepare a mother liquor, and store in a -80°C refrigerator. When used, dilute with culture medium to the required concentration.

[0098] 3) Experimental method

[0099] Preparation of bacterial solution

[0100] (1) Bacterial recovery: The frozen bacterial solution was taken out from the -80 °C refrigerator, thawed, diluted in a biological safety cabinet, and the bacterial diluent was taken with a loop and streaked on NA medium. The plate was inverted and incubated in a constant temperature incubator at 37 °C for 16-18 h.

[0101] (2) Activation: The single colony obtained by the above plate streaking method was picked up with a loop and placed in a 100 mL broth medium, and placed in a constant temperature shaker (37 °C, 180 rpm) for overnight shaking. This activation step was operated at least twice in succession.

[0102] (3) Preparation of bacterial solution: A single colony was inoculated into MH broth medium and incubated in a constant temperature shaker at 37 °C and 180 rpm / min overnight for 16-18 h. It was transferred to a 2 mL centrifuge tube and centrifuged at 8000 rpm / min, washed 3 times, and resuspended with fresh broth liquid medium. The bacterial solution concentration was adjusted to 0.5 McFarland units (1x108 CFU / mL), and then diluted with broth to the required experimental concentration of 1x105 CFU / mL for standby.

[0103] b Determination of growth curve

[0104] In order to study the growth rate of bacteria, the bacterial solution in the logarithmic growth phase required for the experiment was obtained, and the growth curves of two strains of Acinetobacter baumannii were determined. Single colonies of the two strains were inoculated into conical flasks containing 100 mL broth medium, sealed with sterile filter membranes, and incubated in a constant temperature shaker (37 °C, 180 rpm). Samples were taken at 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, respectively. The absorbance of the bacterial solution at 600 nm was measured using a microplate reader, and three parallel experiments were set. The absorbance value was taken as the vertical coordinate, and the time was taken as the horizontal coordinate. The growth curve of the bacteria was drawn using Origin2016 software.

[0105] c Minimum inhibitory concentration determination

[0106] The prepared bacteria solution in logarithmic growth phase was diluted to 1 x 105 CFU / mL, and 50 μΐ, was added to a 96-well cell culture plate. The mother liquor of the control drug CCCP was diluted to 2560 μΜ, and 50 μΐ, of different concentrations of drug solution (2560, 1280, 640, 320, 160, 80, 40, 20, 10 μΜ) was added by two-fold dilution method, and three parallel groups were set for each concentration. The MH broth medium was set as the negative control, and the bacteria solution was used as the growth control. After incubation at 37°C overnight for 16 h, the turbidity of the broth medium in each group of wells was observed, and the lowest concentration without bacterial growth was the MIC value of CCCP. The MIC values of the candidate inhibitors on 2 strains of Acinetobacter baumannii were detected by the same method, and 1 / 4 x MIC concentration was used as the working concentration of the candidate inhibitors and CCCP.

[0107] 4) Efflux pump inhibition experiment

[0108] The mother liquor of 7 antibiotics was diluted to 2560 μΜ, and 50 μΐ, of different concentrations of different concentrations of antibiotics (2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 μΜ) was added to a 96-well plate by two-fold dilution method, and 50 μΐ, of CCCP and 50 μΐ, of 1 x 105 CFU / mL bacteria solution was added. The final concentration of CCCP in each well was 80 μΜ. The broth medium was set as the negative control, and the bacteria solution was used as the growth control. Three parallel groups were set for each group to ensure the accuracy of the results, and the results were observed after incubation at 37°C overnight for 16 h. Similarly, the efflux pump inhibition experiment of the candidate inhibitors was completed by the above method. The results were judged according to the CLSI M100 version standard. When the efflux pump inhibitor CCCP was added, the MIC value was reduced by 4 times or more, which was judged as efflux pump phenotype positive. It is worth noting that the MIC test and the efflux pump inhibition experiment were carried out at the same time.

[0109] 5) Cytotoxicity experiment

[0110] Cytotoxicity experiment is to detect the effect of compounds on the proliferation of normal cells, which can effectively reduce the side effects of drugs in vivo. In this study, CCK-8 detection method was used to evaluate the cytotoxicity of candidate inhibitors, and human normal epidermal fibroblast cell line was used. The specific experimental steps are as follows:

[0111] (1) Cell recovery

[0112] Take out the HSF cell strain in the liquid nitrogen tank, and place it in the cell interstitial water bath pot at 37°C for rapid thawing, that is, all melt within 1 min, and the bottle opening is sprayed with 75% alcohol and placed in the clean bench. The cell suspension in the cryopreserved tube is transferred to a 10 mL centrifuge tube, and an appropriate amount of DMEM medium (about 3-6 mL) is added. The rotation speed is set to 1000 rpm, and after centrifugation for 5 min, the supernatant is discarded, and an appropriate amount of DMEM complete medium is added. The cells are gently blown and mixed evenly, transferred to a culture bottle, and after drawing an 8-character gently mix the cell liquid, spray 75% alcohol, and place in a CO2 incubator at 37°C for culture. After the cells adhere, replace the cell culture medium according to the above method.

[0113] (2) Cell passage

[0114] After the cell coverage in the culture bottle reaches more than 80%, passaging is performed. Discard the excess culture medium in the culture bottle; add 3 mL of PBS to the culture bottle, gently shake for about 30 s, and then discard. Add 2 mL of trypsin and place in the incubator for about 3 min. Under a microscope, the cells have all become round, gently tap the bottom of the culture bottle, and the cells can be clearly seen to disperse. Add 4 mL of culture medium to terminate digestion, and gently blow and mix the bottom of the bottle with a gun head, inoculate in 3 culture dishes containing 7 mL of fresh culture medium, and place back in the incubator (37°C, 5% CO2) for continued culture.

[0115] (3) Cell cryopreservation

[0116] After the cell coverage in the culture bottle reaches more than 80%, and no passaging or experiment is needed. The cells can be washed with PBS, trypsinized, centrifuged, and 1 mL of cell cryopreservation solution is added and transferred to a 1 mL cryopreserved tube for cryopreservation. After overnight storage at -80°C, it is stored in a liquid nitrogen tank.

[0117] (4) CCK-8 method detection:

[0118] After the cells were cultured to the 3rd passage by the above method, they were washed with PBS, trypsinized, centrifuged, and resuspended in 2 mL of DMEM complete medium to prepare a cell suspension with a density of 5 x 104 / mL. First, 90 μL of the cell suspension was added to each well of a 96-well plate, which was then placed in a constant-temperature incubator (37°C, 5% CO2) for 12 h of culture; 10 μL of a series of different concentrations (10, 20, 40, 80, 160, 320 μM) of drug solution was added to each well, and an equal volume of DMEM complete medium was added as a control group. After incubation in the incubator for 10 h, 10 μL of CCK-8 solution was added to each well, and the incubation was continued in the incubator for 2 h. The OD450 value was measured using a multifunctional enzyme marker. Three parallel groups were set up for each group, and the blank group contained only DMEM complete medium and CCK-8 solution. No drug was added to the positive control wells, and an equal volume of DMEM complete medium was added. The inhibition rate of the candidate inhibitor on cells was calculated using the following formula:

[0119]

[0120] All data in the experiment were repeated multiple times, and statistical analysis was performed using GraphPad Prism 9.0 software. When the difference was significant, P<0.05, indicating that the difference was statistically significant (P<0.05, marked with “*”; 0.05

[0121] Experimental results:

[0122] 1) Growth curve

[0123] The growth curves of Acinetobacter baumannii and multi-drug resistant Acinetobacter baumannii were measured using an enzyme marker at a wavelength of OD 600 nm. The experimental results are shown in Figure 4 The growth trends of the two strains were basically the same, with a growth lag phase of 1-3 h and a logarithmic growth phase after 4 h. The logarithmic phase of the bacteria was usually used in experiments to ensure the reliability of the experimental results. Therefore, the bacteria cultured for 12-16 h were selected for subsequent experimental research.

[0124] 2) Minimum inhibitory concentration

[0125] The MIC values of the candidate compounds against A. baumannii and MDR A. baumannii were quantitatively determined by microdilution method, and the results are shown in Table 4. The MIC value of CCCP against the two strains was 320 μM, the MIC value of ChemDiv L676-2179, ChemDiv L676-1461 and Chembridge 53717615 against the two strains was 640 μM, and the MIC value of other compounds was greater than 640 μM. The working concentration of three compounds was determined to be 160 μM, and the working concentration of CCCP was 80 μM. At the same time, the drug sensitivity of the three candidate compounds was evaluated by the drug sensitivity paper method, and the results are shown in Table 6. Figure 5 At 3 times the working concentration, ChemDiv L676-2179, ChemDiv L676-1461 and Chembridge 53717615 did not produce obvious inhibition zones, which met the requirements of efflux pump inhibitors.

[0126] Table 4 Minimum inhibitory concentration determination results

[0127]

[0128] 3) Efflux pump inhibition experiment

[0129] Seven antibiotics with different mechanisms of action, imipenem, cephradine, levofloxacin, amikacin, azithromycin, tetracycline and gentamicin, were selected for efflux inhibition experiments with compound Chembridge 53717615, and the results are shown in Table 5. For A. baumannii, the combination of compound Chembridge 53717615 and levofloxacin, amikacin, azithromycin, tetracycline and gentamicin showed positive efflux phenotype, while the combination of imipenem and cephradine showed negative efflux phenotype. For MDR A. baumannii, tetracycline, azithromycin and gentamicin: Chembridge 53717615 showed positive efflux phenotype for MDR strains, and was much better than the control group CCCP. In summary, Chembridge 53717615 showed different degrees of efflux effect on antibiotics with different resistance mechanisms.

[0130] Table 5 Activity determination of candidate compounds on efflux inhibition of A. baumannii sensitive strain and MDR strain

[0131]

[0132] Note: IPM: Imipenem, RAD: Cephradine, LEV: Levofloxacin, AMK: Amikacin, TC: Tetracycline, AZM: Azithromycin, and GM: gentamicin

[0133] 4) Cytotoxicity

[0134] The CCK-8 method was used to explore the toxic effects of the three candidate compounds on normal cells, and the cell strain was human normal epidermal fibroblasts, and the research results are shown in Table 4. Figure 6 Overall, with the increase of the concentration of the compounds, the cell inhibition effect also showed an upward trend. The cell survival rates of compounds ChemDiv L676-2179 and ChemDiv L676-1461 were greater than 70% at low concentrations (10-40 μM), indicating that the cytotoxicity of the compounds at this concentration was small, better than the control group CCCP; at a high concentration of 160 μM, the cell survival rate was between 10% and 30%, showing a toxicity effect comparable to CCCP. At this concentration, the cell survival rate of the control group CCCP was 37.85%. Chembridge 53717615 had a promoting effect on cell proliferation at low concentrations of 10 and 20 μM; and at a high concentration of 160 μM, the cell survival rate was still above 80%, indicating that it had little effect on cell growth and was of high drug safety.

[0135] The present application is not limited to the above-mentioned embodiments, and those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. Use of Chembridge 53717615, of formula: ###0001### in the preparation of an inhibitor of the Acinetobacter baumannii AdeABC efflux pump. 19 H 25 N5O2 The structural formula is: .

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