Identification Of Anti-bacteria Agents Disrupting The Interaction Between LptA And LptC

Objectives A yeast two hybrid model was used to screen the inhibitors of LptA/LptC interaction in E.coli.The antibacterial activity and mechanism of the positive compounds were further studied.With this research,the new lead antibacterial compounds targeting LptA/LptC interaction were selected and the foundation of the subsequent discovery of antibacterial drugs from microorganisms was laid.At the same time,in vitro screening methods of LptA/LptC interaction based on Biolayer Interferometry（BLI）were established to complement the screening methods based on the yeast two-hybrid model.Methods The plasmids of pAD-LptA and pBD-LptC were extracted and co-transferred into AH109 cells and the expression of reporter genes in AH109 cells were further detected to get AH109（pAD-LptA+pBD-LptC）.The fermentation broth of actinomycetes of our school and the compound library of the National New Drug（Microbial）Screening Laboratory were screened with this model,and AH109（pAD-T+pBD-53）was used as negative control.The compounds showed specific growth inhibitory activity to AH109（pAD-LptA+pBD-LptC）were selected as positive compounds.The antibacterial activities aganist E.coli of these compounds were further evaluated,and the compounds with good activity were further studied.Firstly,the inhibition ofβ-galactosidase expression was detected on the yeast two hybrid systems.Then,LptA and LptC proteins were expressed and purified,and the blocking of the interaction between LptA and LptC was detected by Surface Plasmon Resonance（SPR）technology.The changes of E.coli morphology and membrane structure,the accumulation of LPS in periplasmic space and outer membrane defect induced by the positive compound were detected,and the blocking of LptA/LptC interaction was comprehensively evaluated at the cellular level.The antibacterial and bactericidal activity of the compounds were evaluated.By detecting the sensitivity of the compound to the overexpression strain,we further evaluated whether the antibacterial activity of the compound was related to the blocking of LptA/LptC interaction.The toxicity and pharmacokinetic characteristics of the compound were detected to evaluate the feasibility of the compounds as drug leaders.The method of LptA/LptC interaction evaluation based on BLI technology was established,and the blocking activity evaluation method was preliminarily established using the reported blocker IMB-881.Results 1 A total of 533 actinomycete fermentation broth and 100000 compounds were screened and 37 actinomycete fermentation broth and 19 positive compounds were selected.Among actinomycete fermentation broth,3 fermentation broth showed good antibacterial activities aganist E.coli,which provided the basis for the separation of antibacterial lead compounds from subsequent microorganisms.For positive compounds,a compound named IMB-0042 showed good activity and was selected to study the follow-up activity and antibacterial mechanism.2 At the molecular level,IMB-0042 blocked the interaction of LptA/LptC by binding to LptA protein.For the E.coli cells treated with IMB-0042,the morphology of cells changed from short rod to long rod,the outer membrane was damaged,and the content of LPS in periplasmic space increased.3 IMB-0042 had good antibacterial activity against E.coli ATCC 25922 with MIC of 6.25μg/ml.The compound showed antibacterial activity at low concentration（2～4×MIC）and bactericidal activity at high concentration（8～16×MIC）.4 The IC₅₀ of IMB-0042 on 293T cells was more than 50μM and the acute toxicity test showed that the mice did not die at the concentration of 200 mg/kg.The preliminary pharmacokinetics test showed that the oral bioavailability of the compound was 0.36%,the half-life was 1.72 h,and the intravenous half-life was 1.28 h.5 The LptA/LptC interaction method based on BLI technology was established,and an assessment method of blocking activity was established through compound IMB-881.Through this method,it was found that IMB-881 could bind to LptA to block the interaction,which was consistent with the reported results.Conclusions 1 IMB-0042 can block the interaction of LptA/LptC at molecular and cellular levels,and showed good antibacterial activity against E.coli.Its antibacterial activity is related to the blocking activity of LptA/LptC interaction.2.The toxicity of IMB-0042 is low,but its bioavailability in vivo is low,and its half-life is relatively fast.Its antibacterial activity in vivo needs further evaluation.3 A preliminary screening method of LptA/LptC interaction blocker based on BLI technology was established,which laid a foundation for the screening of molecular level blockers.4 Microbial secondary metabolites are an important source of antibiotics.In this study,LptA/LptC interaction model was used for the first time to screen microbial secondary metabolites,and three strains with antimodel activity and antibacterial activity were obtained,providing a new idea for looking for new anti-Gram-negative bacteria antibiotics targeting the outer membrane.Figure 28;Table 11;Reference 68...