Discovery And Antibacterial Study Of Potential PPK1 Inhibitors Against Uropathogenic Escherichia Coli

Background and objective:Treatment of bacterial infections become more and more difficult because bacteria develop resistance to antibiotics at an alarming speed.Urinary tract infections?UTIs?are one of the most common infectious diseases in clinical,especially among women,the elderly and children.With the worldwide dissemination of drug resistant bacterial strains,clinical treatment of UTIs becomes a serious challenge.Although either Gram-positive or-negative bacteria can cause UTIs,more than 80%of community-acquired UTIs are caused by uropathogenicEscherichia coli?UPEC?.Antimicrobial susceptibility tests indicate that UPEC is resistant to conventional antibiotics such as furadantin and even to the last-line of defenders like colistin.Therefore,the development of new types of antibacterial agents or approaches to combat UPEC is urgently needed.Most of the conventional antibiotics target essential bacterial cell functions.For examples,methicillin and vancomycin target bacterial cell wall synthesis,polymyxins target cell membrane,and quinolones inhibit the critical enzymes of bacterial growth process.However,the resistance to most of these drugs has been observed to date.To reduce bacterial virulence instead of killing the pathogen seems to be an alternative strategy to treat bacterial infections.Poly phosphate kinase 1?PPK1?is an essential kinase in bacterial pathogens and is responsible for the synthesis of poly phosphate?poly P?and metabolism of ATP.Previous studies revealed that PPK1 is essential for bacterial motility,biofilm formation,quorum sensing,and virulence factor expression and is also an important target for the investigation of antibiotics resistance and pathogenicity.Our previous study showed that,in the ppk1 knockout mutant of E.coli,not only the amount of poly P but also the abilities of adhesion and invasion were significantly decreased comparing with the wild type E.coli.In addition,poor survival rates during oxidative stress,osmotic shock and acidic challenge also were found in this?ppk1 strain.In addition,it is reported that PPK1 homologue is absent in human beings.Therefore,PPK1 or its coding gene can become a potential drug target for development of novel antibacterial agents.Over the past decade,only a few compounds were reported to interact with PPK1of E.coli.However,there is no further in-depth study has been reported thus far.Therefore,in this study,PPK1 inhibitors will be selected through virtual screening,and their effects on the pathogenicity of UPEC will be studied through biological experiments and animal experiments to provide experimental evidence for the development of novel antibacterial agents for UTIs.Methods:1.The virtual screening workflow was carried out to identify novel inhibitors ofPPK1 by using several modules embedded in Schr?dinger Suite 2019.Surfaceplasmon resonance?SPR?assay was performed to rapidly screen candidates byevaluating the binding responses between PPK1 and the compounds.Then,tofurther investigate the interaction between the compounds and PPK1,SPR assaywas used to detect the binding affinity of the compounds with PPK1.Andmolecular docking study was conducted to have a better understanding ofcompounds interacting with PPK1.2.4',6-Diamidino-2-phenylxylene?DAPI?staining method was used to determinewhether compounds can affect the activity of PPK1 or not.3.The bacterial growth curve was detected to study whether compounds can affectthe growth of CFT073 or not.4.Ivasion assays were performed to evaluate the impact of compounds compoundson the ability of CFT073 to invade to uroepithelial cells.5.Oxidative stress resistance assay was used to determine the sensitivity to _H2O22O2 ofbacteria.6.Biofilm inhibition assay was conducted to know whether compounds affect thebiofilm formation of CFT073 or not.7.Tthe compounds were determined for its ability to reduce UPEC infection invivoby mouse model experiments.Results:1.Through virtual screening and SPR assay,compounds 8 and 17 were selected tofurther investigation.SPR assay showed that the response values of unit molecularweights of compounds 8 and 17 were 4.073 100RU/Da and 6.555 100RU/Da,respectively.And the dissociation constant?K_D?was 85.8?M and 84.6?M,respectively.2.The results of PPK1 activity assay showed that the activity of PPK1 in thecompounds 8 and 17 treatment groups was reduced?P<0.05?compared with thecontrol group.3.As shown in the results of bacterial growth curve,the growth of CFT073 in thecompounds 8 and 17 treatment groups was not significantly different from that inthe control group.4.The results of ivasion assays and oxidative stress resistance assay showed that,theinvasion and anti-oxidation stress ability of CFT073 in the compounds 8 and 17treatment groups was reduced?P<0.05?compared with the control group.And theresults of biofilm formation assay showed that,compared with the control group,the biofilm formation ability of the CFT073 in the compounds 8 and 17 treatmentgroups was weakened as compared to the control.5.The results of animal efficacy study showed that,the bacterial burdens in thebladder of the mice treated with compounds 8 and 17 was significantly reduced?P<0.05?as compared to the control.Furthermore,from the results of morphologicalexamination,inflammatory cells and the tissue edema and epithelial damage weredecreased in the compound-treated group.Conclusions:1.The results of binding assay and molecular docking study proved that compounds8 and 17 can interact with PPK1.2.Compounds 8 and 17 can effectively reduce the activity of PPK1.3.Compounds 8 and 17 can effectively reduce the invasive ability and resistance tooxidative stress of UPEC?CFT073?without any inhibitory effect on the growth ofthis strain.In addition,in the CFT073 infectious mice model,8 and 17 were foundto reduce bacterial burdens in the bladder and prevent urinary tract infection.4.Compounds 8 and 17can inhibit the biofilm formation of CFT073.5.PPK1 inhibitors have the potential to become novel antibacterial agents to thetreatment of UPEC infection.