Antibacterial Effect Research Of Novel Imidazole Componds For The Target Of WALK Enzyme HATPase-c Domain From Streptococcus Pneumoniae

Objective: Our research team used the target of Wal K enzyme HATPase-c domain from Streptococcus pneumoniae to screen compound 6,and it had significant antibacterial effect,but its body protection effect is not idea.Our research team made six compounds by origin compound 6,and use three-dimensional structure of the amino acid residues as the drug target.Through the interaction of compounds and three different Wal K protein enzyme to validate that the antibacterial target of new compound is in Wal K enzyme CA domain.Through experiment examines its antibacterial activity in vitro and toxic effects.By establishing the model of the infection such as pneumonia to testify the antibacterial effects of in vivo.This study intends to eventually get imidazoles derivative compounds that post low toxicity and high antibacterial activity.Methods: This topic proposed by molecular design,synthesis,biological activity test mode.Remolding compound 6 by reactive joining together and substitution method.The character of new derivatives were surveyed by elemental analysis,1H NMR,13 C NMR and MS(ESI).Using the method of PCR amplification to construct 3 different sequences of Wal K gene(full length,defect in CA domain,CA domain only)which were cloned into pp SUMO plasmid,transformed into E.coli BL21(DE3)and induced by IPTG protein expression.Then using Ni-NTA to obtain high purity protein.The purified recombinant protein was immunized with BALB/C mice to prepare polyclonal antibody,and the specific Ig G titer was detected by ELISA method.Then Wal K full-length protein was analyzed conservative in 4 of the domestic common S.pn serotype strains by western blot.Using ATP Kinase luminescence detection kit to detect the protein kinase activity of three kinds of protein and protein kinase activity after interaction of the new type of imidazoles derivatives and protein,and IC50 is calculated.Through direct action of bacteria and new imidazole derivatives,then calculate the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)to detect the antibacterial effect of derivative.Through the cell toxicity test in vitro to test the toxicity of new imidazoles derivatives.Using S.pn to build abdominal infection model of BALB/C mice,then observe the survival time of mice and survival rate after drug effect.new imidazoles compounds.The Wal K gene(full length,defect in CA domain,CA domain only)was successful constructed and expressed.Recombinant protein was immunized in mice then get high titer polyclonal antibody,Western Blotting analysis confirmed that CMCC(B)31207,CMCC(B)31203,CMCC(B)31693,NTCC7466(D39)S.pn strains could express full-length Wal K protein.As the new imidazoles compound(compound 2,compound 4 and compound 5)concentration increased gradually,ATP kinase activity of the Wal K full length and CA domain continuously decreased(P<0.05)and the ATP kinase activity of deficient protein has no difference between the change(P>0.05).Compound 2,compound 4 and compound 5 have good antibacterial properties,compound4 posed the most significant bacteriostatic effect(P<0.01).Cell toxicity test(determined by MTT)confirmed that the three compounds cytotoxicity were significantly lower than the original compound(P<0.01),with compound 2 owing the lowest cell toxicity(1170?M).The treatment effect of Compounds to mice that infected streptococcus pneumonia D39 confirmed that compared with positive control group(PNC)the original compounds 6,new synthetic imidazoles compound 4 and compound 5 has no obvious difference on protective effect.Compound 2 owned obviously difference with positive control group(P<0.05).Results: We cooperated with zunyi medical college of pharmacy.Preliminary getting sixConclusion: Wal K full-length recombinant protein was conservative in different serotype S.pn.Through the experiment proves the target of the new imidazoles derivatives(compound 2,compound 4 and compound 5)located in Wal K kinase CA domain.Compound 4 have good antibacterial properties by MIC and MBC.The cytotoxicity of new compound2 was the lowest.Compounds 4,compound 5 have significantly protective effect in the mice that infected streptococcus pneumonia D39.This study is the first time to show the new synthesis imidazoles compound 4 may be the future treatment of streptococcus pneumoniae infection.Follow-up study will focus on the in-depth study of drug metabolism and pharmacokinetics,so as to screen out broadspectrum imidazoles new compounds to resist gram-positive bacteria infection.