| Bacterial resistance has become an imminent global problem.Recent studies have found that hydrogen sulfide(H2S)-producing enzymes in bacteria are closely related to microbial survival and antibiotic resistance.In addition,bacterial H2S is known to be involved in many physiological and pathological processes of infectious diseases.In bacteria,the production of H2S mainly depends on 3-mercaptopyruvate sulfurtransferase(MST),cystathionine ?-synthase(CBS)or cystathionine y-lyase(CSE).However,no specific and active inhibitors have been found for these bacterial H2S enzymes.Among them,MST is the only H2S producing enzyme present in Gram-negative bacteria.At present,more and more Gram-negative bacteria including E.coli and Klebsiella pneumoniae strains have developed strong drug resistance.Thus,finding new inhibitors of bacterial MST could advance new antibiotics or antibiotic synergists and could establish the drugability of MST-based agents to overcome bacterial resistance.The main work of this thesis is as follows:(1)A detailed study on the enzymatic activity of E.coli MST(eMST)has been conducted.The enzymatic activity was studied under different buffers,pH and enzyme concentration conditions using 192-tandem plates to perform the real-time detection of enzyme activity of eMST.The optimal enzyme activity conditions were determined(Hepes buffer,pH 7.2;1 mM 3-mercaptopyruvate(3-MP)and 2 mM homocysteine(Hcys);38.5 nM eMST).At the same time,the enzyme kinetic constants of eMST were studied in detail,and it was found that the Km value of the substrate 3-MP is similar to that of human MST(hMST),but the catalytic constant kcat exceeds more than 1000 times than that of hMST,suggesting that eMST is a much more efficient enzyme.In addition,we also systematically studied and compared two different activities of eMST,i.e.3-MP substrate-based transferase activity and thiosulfate-substrate-based rhodanese activity and validated that the enzyme has the functions of these two types of activity.(2)We developed a high-throughput enzyme activity assay method for eMST and screened?26,320 compounds from different compound libraries using this method.Menadione(a known hMST inhibitor)was used as an MST inhibitor to verify the reliability of this high-throughput assay.Subsequently,two thiazolidinediones(pioglitazone and rosiglitazone)and some natural products were identified as specific inhibitors of eMST(IC50?20 ?M).(3)Several specific and selective inhibitors of eMST,identified by the high-throughput assay,were characterized.First,the selectivity of the compounds was identified by testing the activity of eMST inhibitors on hMST,hCBS,and hCSE.It was found that thiazolidinedione inhibitors did not inhibit the activity of these homologous enzymes,indicating that the compounds are selective inhibitors of eMST.At the same time,the specificity of inhibitors was verified using interference assays(including redox assay,non-enzymatic release of H2S interference experiments,substrate 3-MP accumulation,etc.).It is particularly important to determine the specificity of the inhibitor because eMST is very susceptible to ROS and non-specific compounds will oxidize the enzyme or bind to the product H2S,resulting in false inhibition signals.(4)We studied the molecular mechanism and mode of action for these thiazolidinediones inhibitors.Through surface plasmon resonance(SPR),enzyme kinetics,molecular modeling,and mutant experiments,these types of compounds were found to directly bind to eMST.Also,it was determined that these inhibitors bind to the enzyme in a reversible manner,competing with the substrate 3-MP,but not with the co-substrate Hcys.Furthermore,two inhibitors were found to selectively bind to Arg178 and Ser239 residues in the active site of eMST.Evidence from molecular modeling studies is presented,which indicates two pockets in the active site of eMST and these pockets can bind the thiazolidinedione head and pyridine tail moiety of the inhibitors.Interestingly,the pocket binding to the pyridine tail does not exist in the hMST structure,suggesting that this structural difference may be responsible for the compound' s selectivity for eMST.(5)We studied the cellular activity of specific eMST inhibitors at the bacterial level and used this to study the mechanism by which eMST regulates H2S production in living bacteria.It was found that pioglitazone can result in the accumulation of 3-MP substrate in bacteria and reduce H2S content,suggesting that it inhibits eMST activity in bacteria.Furthermore,using a novel reactive oxygen probe roGFP2,it was confirmed that pioglitazone can enhance the production of reactive oxygen species(ROS)in bacteria.Later,the bacteria-killing effects from combined pioglitazone and gentamicin was studied,and it was found that pioglitazone enhances the lethality of gentamicin against E.coli.Additionally,it was shown that the addition of exogenous H2S could prevent this synergistic effect.However,in the inhibitor-insensitive eMST mutants(R178L,S329A or C237A)or eMST knockout bacteria,pioglitazone has no regulatory effect on substrate content,suggesting that it has an on-target effect for eMST in bacteria.Finally,in a Raw264.7 macrophage-based E.coli infection model,pioglitazone demonstrated a significant bactericidal effect,suggesting that it has the potential to become an antibiotic synergist.In conclusion,this thesis demonstrates pioglitazone as the first specific and selective inhibitor of eMST and confirms that it can target and regulate eMST in vitro and in living bacteria.This regulatory effect has also been shown to be able to enhance the antibacterial effect of antibiotics.Using pioglitazone as a small molecule tool,the activity and function of eMST could be studied in other E.coli pathogens,Gram-negative bacteria and in the process of animal infections,to confirm its drugability during infection,and could provide a new type of antibiotic synergist for the treatment of related diseases. |
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