Candidate Drug Development And Target Discovery For Anti-drug-Resistance Staphylococcus Aureus By Targeting CrtN

Staphylococcus aureus(S.aureus)is a major pathogen that causes severe skin soft tissue and respiratory system infections and more serious illnesses such as pneumonia,endocarditis and sepsis.Recently,under the abuse of antibiotic,S.aureus has developed severe resistance.Multi-drug resistant methicillin-resistant S.aureus(MRSA)has emerged,seriously endangering human life and health.The mechanism of traditional antibiotics can not solve the severe drug resistance.In particular,there is an urgent need to develop new drugs that can not only effectively treat the infections of MRSA but also avoid mutations.The virulence factors secreted by bacteria have become important targets,because they can not only create a more suitable growth and reproduction environment for bacteria in the host,but also enable bacteria to escape from the immune system.The inhibition of bacterial virulence factors enables the host to exert his own autoimmune ability(such as ROS killing)without affecting the normal growth of the bacteria,effectively avoiding bacterial resistance.The golden carotenoid pigment(staphyloxanthin)is an important virulence factor that shields S.aureus from host ROS killing,so the catalytic enzymes involving in the staphyloxanthin biosynthesis are underlying magnetic anti-virulence targets for the inhibition of S.aureus.The previous studies in our laboratory found that the antifungal old drug naftifine and its derivatives can effectively inhibit the growth of S.aureus by inhibiting the key catalytic enzyme CrtN and showed good efficacy in animal-infection model.In this study,the benzofuran derivative 1 and the benzocycloalkane derivative 67,which were discovered by us,were lead compounds.Keeping the good efficacy in vivo and in vitro,we synthesized analogues which could reduce the hERG cardiotoxicity and increase the antibacterial efficacy.We aimed to develope a candidate with excellent drug properties and market competition.In the 2nd chapter,taking 1 as the lead compound,we synthesized compounds 2a-d,and 2b was taken as the preferred maternal structure according to pigment inhibitoryactivity of Newman.Through structural optimization of three different regions(A-C),aclear structure-activity relationship was summarized and compound 23 was confirmed tobe the candidate structure,along with the cytotoxicity of HEK-293 and HepG-2.Compound 23 also inhibited drug-resistant S.aureus(USA400 MW2,USA300 LAC,Mu50 and NRS271)at the nanomolar level.For the most critical hERG toxicity,compound 23(ICso = 3.71 ?M)outperformed lead compound 1(IC50 = 34.8 ?M),solving the priority problem of our former work.Compound 23 inhibited CrtN at the nanomolar level too and did not affect bacterial growth at high concentrations(200 ?M),furthermore it also lost antifungal activity(MIC>8 ?g/mL).Compound 23 demonstrated good stability(T1/2 ?50.7 mins)in human liver microsomal.The experiments of H2O2 and human whole blood killing showed that compound 23 could make S.aureus more susceptible to oxidative damage.In animal parts,compound 23 showed good antimicrobial efficacy in both pre?infection and post-infection administration modes,significantly reducing Newman and resistant bacterias(Mu50,NRS271,LRSA56,LRSA202,NF65Y and XN108)in kidneys and hearts(bacteriostatic rates>95%)of mice.Compound 23 was also effective in prolonging survival in sepsis model.Nine other strains of MRSA were tested for their in vitro pigment inhibitory activity(comparable to inhibitory activity of Newman),demonstrating that compound 23 could inhibit S.aureus with broad spectrum.For eight subtypes of CYP enzyme,basically,compound 23 showed no inhibitory activity.Compound 23 showed superior oral bioavailability(83.8%)in mice in the pharmacokinetics experiments.Finally,we found that the candidate compound 23 did not induce drug resistance and the probability of bacterial mutation was less than?1 × 10-5 after 12 generations with the stress of oxidation and drug.In the 3rd chapter,compound 67,which was reported in our earlier study,was also the lead compound.We increased the number of oxygen atoms in benzocycloaliphatic and found compound 68 showed best inhibitory activity of Newman.A total of 58 derivatives were designed,synthesized and evaluated by the pigment inhibitory activity for Newman.A clear structure-activity relationship was obtained.Analyzing a series of in vitro druggable evaluation,compounds 112 and 115 were confirmed as candidate compounds.Immune clearance experiments and bacterial growth tests demonstrated that 112 and 115 were able to make S.aureus more susceptible to oxidative damage by H2O2 and human whole blood without affecting the normal growth of bacteria.Compounds 112 and 115 also showed significant antibacterial activity and broad spectrum(S.aureus Newman,Mu50 and NRS271).In the animal part,compounds 112 and 115 showed comparable antibacterial activity to VAN and LZD in livers and hearts(bacteriostatic rates>95%).For MRSA strains(Mu50 and NRS271),both compounds 112 and 115 surpassed VAN or LZD.Among the sepsis models administered by lethal dose of Newman,compounds 112 and 115 were also effective in prolonging the survival of mice.Based on the results,the structural optimization of lead compounds 1 and 67 was successful,not only maintaining potent in vitro and in vivo antibacterial activity but also greatly solving the potential cardiotoxicity of hERG inhibition.In conclusion,we finished a very comprehensive druggable evaluation for the candidate compound 23,which improved the follow-up clinical development.The innovations are summarized as:(1)Completely overcome the lack of hERG cardiotoxicity in the previous studies of benzofuran and benzocycloalkane derivatives;(2)The animal part,different administration methods,doses,and multidrug-resistant bacterial infections were increased,and the efficacy of the candidate compound was confirmed in all aspects;(3)The in vtiro pigment inhibitory activity of 1 wild strain of Newman and 13 strains of MRSA were tested,proving the broad spectrum of the inhibition of S.aureus.(4)Verification of drug metabolism was performed in a variety of forms of administration including intravenous injection,oral and intraperitoneal injection,and the results of acute toxicity were verified;(5)Inspected the results of drug resistance,and effectively proved that "bacteriostatic,not bactericidal" can effectively reduce the concept of resistance mutations.