Discovery And Activity Study Of Inhibitors Of The Interaction Between The Fission Protein FtsZ And SepF Of Mycobacterium Tuberculosi

Tuberculosis(Tuberculosis,TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(M.tuberculosis,Mtb),which presents a significant public health challenge.In 2021,the number of people developed TB infection in China was about 780,000.During the COVID-19 epidemic,the number of people who developed MDR-TB or RR-TB(MDR/RR-TB)has increased,making TB treatment more difficult.Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens.Therefore,new anti-TB agents working by different mechanisms are urgently needed.FtsZ,a tubulin-like protein with GTPase activity,forms a dynamic Z-ring in cell division.In Mtb,the function of Z-ring is modulated by SepF,a FtsZ binding protein.The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division.Here,we established a yeast two-hybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M.tuberculosis.Firstly,the interaction between FtsZ and SepF was confirmed by yeast two-hybrid system.Then,based on this,a high-throughput screening model for FtsZ/SepF interaction inhibitors was developed.The disruption of FtsZ/SepF interaction by small molecules or drugs would prevent the growth of yeast in SD/-Ade-His medium,or inhibit the β-gal activity.About 10,000 compounds from National New Drug(Microbial)Screening Laboratory Compound Library were screened and FtsZ/SepF interaction inhibitor T0349 was obtained.In order to investigate the mechanism of T0349,FtsZ and SepF proteins from M tuberculosis were expressed and purified in E.coli.We have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down,fluorescence polarization(FP)and surface plasmon resonance(SPR).In addition,the CRISPRi system was used to identify the target protein of T0349.Mycobacterium smegmatis was used as an alternative strain to get the gene knockdown strains and protein overexpression strains.By susceptibility test,we demonstrated that compound T0349 disrupts FtsZ/SepF interaction by binding to SepF.The anti-Mtb activity of compound T0349 was determined.The MICs of T0349 were varied from 1 μg/mL to 4 μg/mL,which was comparable to the first-line anti-Mtb drug rifampicin.Moreover,T0349 had no significant activity against other bacterial strains.T0349 showed selective activity against M.tuberculosis.In summary,we established a yeast two-hybrid based screening system for the inhibitors of FtsZ/SepF interaction in M.tuberculosis.And using this system,we obtained the FtsZ/SepF interaction inhibitor T0349.Further experiments demonstrated that compound T0349 blocked the FtsZ/SepF interaction by binding to SepF.Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.