| Staphylococcus aureus is an important human pathogen.With the emergence of multidrug resistance,there is urgent need to develop new agents(new structures and new mechanisms)for treatment of this pathogen.The golden carotenoid pigment(staphyloxanthin)is an important virulence factor that shields S.aureus from host oxidant killing,so the catalytic enzymes involving in the staphyloxanthin biosynthesis are underlying magnetic anti-virulence targets.We have firstly built the phenotypic screening model for the pigment inhibition,and screened an in-house collection composed of commercially available known drugs for inhibitors of the staphyloxanthin biosynthesis of S.aureus Newman.Of the 412 drugs tested,an old anti-fungal drug naftifine hydrochloride was found to be an effective staphyloxanthin biosynthesis inhibitor(IC50=296 nM),and sensitized S.aureus Newman to H2O2 or human whole blood clearance.The addition of naftifine to staphylococcal cultures(up to 0.2 mM)did not affect the growth of S.aureus Newman.Additionally,naftifine was capable of blocking the staphyloxanthin biosynthesis of several MRSA strains(USA300 LAC,USA400 MW2,and Mu50)at low micromolar concentrations.We determined the pigment inhibition was mediated by the competitive inhibition of S.aureus diapophytoene desaturase(CrtN),an essential enzyme for carotenoid pigment synthesis,by series of chemical biology methods.To preliminarily determine the pigment inhibiton activity of naftifine by targeting inhibiton CrtN with certain structure specificity,we designed and synthesized 8 derivatives based on naftifine scaffold,and through the SAR analysis we proved naftifine and its analogues were totally novel and potent CrtN inhibitors.Moreover,we found that nafti fine attenuated the virulence of a variety of clinical,S.aureus isolates,including methicillin-resistant S.aureus(MRSA)strains,in mice infection models.These findings provided proof-of-concept that CrtN was a druggable target against pigmented S.aureus infections,revealing naftifine was an excellent lead compound as CrtN inhibitor.Considering that naftifine was a known drug molecule and its structure had been protected by the original company,this thesis selected naftifine as a lead compound to pursue novel CrtN inhibitors,and scaffold hopping was employed to design and synthesize two new skeleton derivatives,transforming the parent skeleton naphthalene ring to benzofuran and benzocycloalkane ring,respectively.In benzofuran series,we designed and synthesized 33 new benzofuran analogs in total(16～18,A1～3,B1～6 and C1～21)and pigment inhibition assay showed there were 16 analogs(B2,B5,C5～16,C19 and C21)exhibiting powerful activities with nanomolar IC50 values and five of them(B2,C10,C11,C13 and C21)exhibiting extremely strong activities with single digital nanomolar IC50 values.Definite SAR was generated based on the analysis of all the compounds pigment inhibition activities.In benzocycloalkane series,we designed and synthesized 49 new benzocycloalkane derivatives(19～37,D1～3,E1～6 and F1～21)totally.And pigment inhibition assay showed there were 31 analogs(22～24,E2,F5,F7～16,F18～19,F21 and 25～37)exhibiting powerful activities with nanomolar IC50 values and eight of them(F8,F10,F11,25～26,28,30 and 33)exhibiting extremely strong activities with single digital nanomolar IC50 values.Definite SAR was generated based on the analysis of all the compounds pigment inhibition activities and just similar to the benzofuran analogs.According to the research strategies similar to naftifine,we determined that these two skeleton derivatives inhibited the staphyloxanthin biosynthesis by targeting CrtN through the HPLC assay,which was in agreement with naftifine.Taking the pigment inhibition activities and structure diversity into consideration,we carefully selected five benzofuran analogs(C7,C10,C11,C13 and C21)and six benzocycloalkane derivatives(F8,F11,25,28,30 and 33)to test their in vitro enzyme inhibition activities.All of them showed powerful enzymatic activities with nanomolar IC50 values.Further considering the good water solubility,C13 and F8 were chosen to be the candidate drugs for further in vitro and in vivo studies.C13 and F8 could efficiently block the staphyloxanthin biosynthesis of three MRSA strains(USA300 LAC,USA400 MW2,and Mu50)without influence on the growth of S.aureus,proving again that compounds in this thesis exerted antimicrobial efficacies by anti-virulence mechanism.Moreover,C13 and F8 sensitized S.aureus Newman and three MRSAs to H2O2 and human whole blood clearance.In mice systematic infection model,C13 and F8 could significantly lowered the survival of S.aureus Newman and two MRSAs in the host organs superior to BPH-652(an antimicrobial candidate drug targeting CrtM)in all cases.To explore whether the derivatives in this thesis possessed anti-fungal efficacies similar to naftifine.we selected several naftifine sensitive fungi to test the anti-fungal activities of C13 and F8,and the results showed these new candidates abated the anti-fungal activities and performed good selective antibacterial efficacies.This thesis preliminarily evaluated the safety profiles and pharmacokinetics of C13 and F8.As indicated,C13 and F8 both possessed good safety profiles and pharmacokinetics superior to naftifine.Especially,C13 possessed good oral bioavailability(F=42.2%).The innovation points of this thesis focused on the following three aspects:(1)we firstly discovered the new application of the old drug naftifine for anti-Staphylococcus aureus;(2)we firstly provided proof-of-concept that CrtN was a good druggable target against pigmented S.aureus virulence;(3)we firstly developed two novel skeleton CrtN inhibitors,and several of them,revealing excellent in vitro/in vivo efficacies,good safety profiles and pharmacokinetics,can be selected as pre-clinical candidate drugs for further comprehensive development.Some research results of this thesis have achieved technology transfer,and subsequent pre-clinical research is ongoing in the pharmaceutical enterprise. |
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