In Pdf As A Target For New Drug Screening And Active Component Separation And Purification, Identification And Activity Research

With the incorrect use of antibiotics or even abuse,the rapid emergence of various G⁺ resistant bacteria,such as MRSA,MRSE,VRE,presents a very serious threat to public health.Therefore,searching for new targets and developing new drugs with novel mechanisms and potent activity against G⁺ resistant bacteria have become an urgent need and an international research project.Peptide deformylase(PDF) is an essential enzyme in the protein maturation of prokaryote,but it is absent from mammalian cells.As its many advantages as a drug target,in recent years,PDF has been widely regarded as one of ideal targets for the screening of new broad-spectrum antibiotic agents.In this study,PDF of E.faecium was cloned and expressed adopting the methods and techniques of molecular biology.The protein obtained was purified by Ni²⁺ metal chelating affinity chromatography column and was identified by Western blotting and activity assay.The screening targeted on PDF includes two parts:screening for inhibiters against E. faecium and screening for inhibiters against PDF activity.As a result,6 positive strains w -ith stable fermentation broths activity were picked out through the initial screening of 20,261 samples of microbial fermentation broths and the other twice re-screening of positive samples.Among them,fermentation broths of positive strains I03A-00723 and I03A-08772 which were identified as nova species of Actinoplanes and named as Aactinoplanes sichuanensis sp.nov,and Actinoplanes xinjiangensis sp.nov,respectively have antimicrobial activity against G⁺ resistant bacteria including MRSA,MRSE,E. faecalis HH22 and VRE.The fermentation broth of positive strain I06A-01113 which belongs to streptomyces has antimicrobial activity against VRE not MRSA.The positive strain I03A-00723 was fermented largely,its fermentation broth was isolated and active components were purified.As a result,8 components were obtained which were 95-1,95-1-h1,95-2,95-3,135,205-1,205-2 and 205-3.Among them,95-1, 95-2,135 and 205-1 were identified as N^b-acetyltryptamine,daidzein,genistein and adenosine respectively and 95-1-h1,95-3,205-2 and 205-3 haven't been identified because of a little content of their samples.It was the first time that N^b-acetyltryptamine, daidzein and genistein were discovered to be produced by strain of Actinoplanes and to have inhibitory activity not only to enzyme PDF but also to E.faecium.Furthermore,the activity of daidzein and genistein against VRE are superior to a known PDF inhibitor Actinonin,and the activity of the minimal component 95-3 against VRE is superior to positive compounds BB3497 and VRC3375 and is approximate to LBM415 which has been in a stage of clinical study,therefore 95-3 is worthy to be studied further.Virtual screening was done with Surflex-Dock in the syby17.3 software on Microbial Natural Products Database,based on the crystal structure of PDF.Some virtual screening compounds were tested the inhibitory activity to PDF and the antibacterial activity to E.faecium.As a result,PolyoxinB,Spergualin and L-4-oxalysine were discovered to have inhibitory activity to PDF and E.faecium.Meanwhile,positive components 95-1,95-2, 135 and known positive compounds such as Actinonin,LBM415,BB83698,VRC4307, VRC3375 and BB3497 were docked with PDF crystal structure,the results showed that their virtual screening scores are correspond to their activities to PDF and E.faecium.In this study,several active compounds inhibiting PDF were obtained by applying model screening combining with virtual screening and the research results would lay foundation for finding inhibitors or lead compounds targeted on PDF.