Screening,Activity Evaluation And Interaction Mechanism Of Inhibitors Targeting New Delhi Metallo-β-lactamase-1

Escherichia coli（E.coli）is not only commensal bacteria in the gut,but also an important gram-negative pathogen of veterinary clinical livestock.The colibacillosis of livestock caused by pathogenic E.coli seriously threatens public health safety and the healthy development of animal husbandry.β-lactam antibiotics have become the first-line drugs commonly used in the treatment of livestock colibacillosis due to their broad spectrum and high efficiency,but with their long-term use in large quantities,resulting in more and more serious drug resistance of E.coli and even multi-drug resistance.Meropenem（MEM）and other carbapenems are known as the“last line of defense”in the clinical treatment of serious infections with multidrug resistant gram-negative bacteria,which have not yet been approved for use in animal husbandry,but a large number of carbapenem-resistant E.coli of animal origin have emerged.The production of New Delhi Metallo-β-lactamase-1（NDM-1）is the most important mechanism of resistance to carbapenems in Enterobacteriaceae.NDM-1 is the most important enzyme of Metallo-β-lactamases（MBLs）,can stably and efficiently hydrolyze almost allβ-lactam antibiotics except monocyclic ones.However,currently clinically applied serineβ-lactamases（Serine-β-lactamases,SBLs）inhibitors have no inhibitory effect on it.NDM-1 has become an important target for screening new drug-resistant enzyme inhibitors due to its broad drug resistance spectrum,multiple variants,and easy transmission through the plasmids.Screening for inhibitors that specifically target the NDM-1 enzyme is the most effective means to inhibit“superbug”infections and protect the effectiveness ofβ-lactam antibiotics,but there is currently no effective inhibitor of NDM-1 enzyme in clinical treatment.Therefore,this study carried out the screening of NDM-1 inhibitors,the evaluation of synergistic antibacterial activity in vitro and in vivo,and the research on the inhibition mechanism,aiming to provide research basis and new strategies for the prevention and treatment of NDM-1“superbug”infection.First,NDM-1 inhibitors were virtually screened based on molecular docking.In this study,by using the technical means of computer-aided drug design and the screening platforms of Glide and Maestro,with NDM-1（PDB:4EY2）as the receptor protein and semi-flexible docking method,the small molecular compounds in the ligand library of Target Mol were molecular docked and scored with the zinc ion(Zn²⁺)active center of NDM-1,respectively.The binding free energies of all docked receptor-ligand complexes were ranked from low to high,and it was found that the Gibbs free energies of the top 100 docked complexes were all less than-9.0 kcal/mol.By analyzing the binding mode of the receptor-ligand complex,it was found that the small molecule compounds were all bound to the Zn²⁺active center of NDM-1,and formed hydrogen bonds,van der waals force,and other various interaction forces with the Zn²⁺and the amino acid residues such as Cys,His,Asp and so on,which greatly increased the affinity of the small molecular compounds with NDM-1.A variety of functional groups and side chain fragments in the small molecule compounds played a key role in the recognition and localization of NDM-1 protein,which initially demonstrated the correctness and reliability of the molecular docking virtual screening procedure for NDM-1 inhibitors used in this experiment.According to the Lipinski drug-like properties,root mean square deviation（RMSD）and ligand efficiency of small molecule compounds,30 candidate inhibitors were further screened.They were spatially matched to the active center of NDM-1 and tightly bound to form a strong interaction force with the high activity efficiency,and have the potential to become effective inhibitors of NDM-1.The NDM-1 enzyme activity inhibition system was established to further screen effective candidate inhibitors and evaluate their antibacterial activity in vitro.The recombinant expression system[E.coli BL21（DE3）-p ET-32a（+）-NDM-1]was constructed by double digestion method,and the NDM-1 recombinant protein with a concentration of about 1.6 mg/m L and a purity of more than90%was obtained by induction of expression and chromatographic purification.The hydrolysis test of Nitrocefin（NIT）showed that compared with the absorbance of the control group without NDM-1,the absorption wavelength of NIT after being hydrolyzed by NDM-1 jumped from 380 nm to 492nm in the visible spectral region after adding NDM-1,and the color of the system also changed from yellow to red,OD492nm increased significantly with time,and the linear relationship was good,indicating that the recombinant protein NDM-1 had a high enzymatic hydrolysis activity.On this basis,the NDM-1 enzyme activity inhibition system was constructed and 30 candidate inhibitors were further screened to obtain four inhibitors with better biological activity,Amifostine（AMI）,Phthalylsulfacetamide（PSA）,Vidofludimus（VFM）and Betaxolol（BET）.The four inhibitors showed highly significant dose-dependent inhibition of NDM-1 enzyme activity（P<0.01）,with the inhibition rates of 83.7%,93.3%,89.2%,and 87.5%,respectively,and the IC₅₀ of 25.4±0.6、13.8±0.5,15.4±0.3 and 19.3±0.9μM,respectively,which indicated that these four inhibitors had significant inhibitory effect on the enzymatic hydrolysis activity of NDM-1 carried by E.coli.The antibacterial activity in vitro of the four inhibitors combined with MEM were determined by minimum inhibitory concentration（MIC）,fractional inhibitory concentration index（FICI）,growth curve and time-kill curve tests.The results showed that the four inhibitors used alone had no significant effect on the growth of NDM-1-positive E.coli,while when combined with MEM was able to reduce the MIC value of MEM against NDM-1-positive E.coli by 4-32 times.The four inhibitors combined with MEM could effectively enhance the antibacterial activity of MEM against NDM-1 positive E.coli,with significant synergistic effects.The FICI were 0.375,0.156,0.125 and0.25,respectively,among which PSA,VFM combined with MEM had the best synergistic effect.In order to further investigate whether the combination of PSA or VFM with MEM has synergistic antibacterial effect in vivo,an NDM-1-positive E.coli infection model was established in BALB/c mice,and the effective dose screening and therapeutic experiments of PSA or VFM combined with MEM were conducted in vivo.Firstly,by comparing and analyzing the white blood cell count,colonization of target organs and inflammatory factor content of mice in different dose treated groups,it was determined that 20 mg/kg PSA and 10 mg/kg VFM combined with MEM had the best effect,and the effective doses had no obvious toxicity to host cells.Subsequently,at the determined effective dose,the efficacy of PSA or VFM combined with MEM in vivo was evaluated through therapeutic studies such as protection rate,white blood cell count,colonization of target organs,determination of inflammatory factor content,histopathological and ultrastructural observation of target organs.The results showed that compared with the infection control group,PSA or VFM single inhibitor group,and MEM single antibiotic group,PSA or VFM combined with MEM significantly improved the survival rate of NDM-1-positive E.coli infected mice（P<0.05）,and reduced the number of NDM-1-positive E.coli colonization in the liver,spleen,and small intestine,and the levels of IL-1β,TNF-α,and IFN-γinflammatory factors in the serum（P<0.05）,and effectively alleviated pathological damage in the liver,spleen and small intestine of target organs of infected mice.This study confirmed that PSA and VFM alone used had no therapeutic effect on NDM-1-positive E.coli infected mice,and MEM alone used had a weak therapeutic effect,while PSA or VFM combined with MEM had a significant synergistic therapeutic effect.In order to explore the inhibitory mechanism of PSA and VFM on NDM-1,we firstly confirmed that PSA and VFM did not affect the expression of NDM-1 by western blotting,and the secondary structure and thermal stability of the NDM-1were not significantly affected by circular dichroism.Secondly,molecular dynamics simulations of the complex system of PSA and VFM with NDM-1showed that the average RMSD of the complex system was only about 2.0?,which revealed that the displacement of the main chain atoms of NDM-1 was small during the dynamics simulation and the complex system could reach dynamic equilibrium in a short time.MM/GBSA binding free energy and free energy decomposition calculations showed that the total binding free energy of the complex system mainly came fromΔE_vdw,PSA and VFM were stably bound to the active hydrolysis center of NDM-1 and interact with Zn²⁺and amino acids.Among them,PSA formed strong interactions with amino acid residues Phe70,Val73,His122 and Ile221,and VFM formed strong interactions with amino acid residues Met67,His120,His122,and His250.Subsequently,in order to verify the results of molecular dynamics simulation,the amino acid residues Val73/His122 and Met67/His120,which contributed significantly to the binding free energy in the PSA,VFM,and NDM-1 complex systems,were site-directed and double-mutated into the inert amino acid Ala.Through the enzyme activity inhibition,fluorescence quenching and molecular docking of wild-type and mutant NDM-1,the results showed that PSA and VFM inhibited wild-type NDM-1 activity significantly stronger than mutant NDM-1（P<0.01）.The binding ability and binding probability of PSA and VFM with wild-type NDM-1 were greater than that of mutant NDM-1,which confirms that PSA and VFM were competitively inhibited hydrolysis activity of NDM-1 enzyme by binding to key amino acids in the active center of NDM-1.Finally,the interaction of PSA,VFM and NDM-1molecules was applied to by Octet RED96 biolayer interferometry,and it was demonstrated that the inhibitors of PSA and VFM could directly bind to NDM-1 to produce interaction with strong affinity.Therefore,the mechanism of action of the small molecule inhibitors PSA and VFM is competitive inhibition.In conclusion,in this study,two novel inhibitors of NDM-1,PSA and VFM,were screened by molecular docking,which could significantly inhibit the hydrolytic activity of NDM-1 enzyme and significantly enhance the antibacterial activity of MEM against NDM-1-positive E.coli.PSA and VFM combined with MEM showed significant synergistic antibacterial effect on NDM-1 positive E.coli in vitro and in vivo,and revealed the mechanism of its competitive inhibition.The above results provide the basis for the development of NDM-1 specific inhibitors targeting“superbug”and new strategies for the prevention and treatment of infections by resistant bacteria.