Screening Of New Antituberculosis Drugs And Preliminary Study Of Their Mechanisms Of Action

Tuberculosis(TB)is a chronic respiratory infection caused by Mycobacterium tuberculosis(Mtb)and is one of the top causes of death among infectious diseases worldwide.According to estimates by the World Health Organization(WHO),one-th ird of the world's population is infected with Mtb.In 1993,the WHO announced that TB entered a global state of emergency due to increased population mobility,the emergence of multidrug-resistant TB(MDR-TB)and extensively drug-resistant TB(XDR-TB).At present,the Bacille Calmette-Guerin(BCG)is the only vaccine used to prevent TB.Although it can prevent severe forms of TB in children,it has no preventive effect on adult TB.TB control is mainly depend on drug treatment.Most importantly,the emergence of MDR-TB and XDR-TB has further complicates the control and treatment of TB for the treatment duration has been lengthened which has complicated its management.In recent years,bedaquiline and delamanid have been approved for TB treatment.However,high toxicity coupled with side effects and high risk limit their use to only adult MDR-TB when no other treatment options are available.As such,development of new antituberculosis drugs with strong bactericidal activity,good safety and effective against clinical drug-resistant TB is urgently needed for control and treatment of TB.In this study,an antituberculosis drug(s)screening model based on the selectable marker-free autoluminescent Mtb H37Ra(UAIRa)was used to screen new compounds for antituberculosis activity and effect in vitro and in vivo,respectively.So as to discover candidate compounds with antituberculosis activity both in vitro and in vivo,and preliminary study the mechanism of action of candidate compounds.The main purpose of the study was to obtain a new compound which has strong antituberculosis activity in vitro and in vivo with little toxicity and side effects.By screening compounds in vitro and in vivo,drug candidates for antituberculosis can be developed to new drugs for clinical treatment of TB which can also facilitate depiction of their core structure thereby laying foundation for development of antituberculosis drugs.The results showed that compound 16j had an MIC of 2.5 ?g/mL in vitro against the UAIRa,and it significantly reduced the bacterial burden in lungs(P<0.001)and spleens(P<0.001)at a dose of 100 mg/kg in the UAIRa rapid-infection mouse model.When compound 16j was combined with SMX,there was partial synergistic activity(FICI=0.74)against UAIRa in vitro.Thus,we inferred that DHFR might be a potential target of the compound 16j.On the other hand,compound 5a had an MIC of 1.25 ?g/mL against the UAIRa and exhibited more significant efficacy in reducing the bacterial burden in lungs(P<0.0001)and spleens(P<0.0001)at a dose of 100 mg/kg in the UAIRa rapid-infection mouse model.Following strong activity in the UAIRa rapid-infection mouse model,compound 5a was tried in Mtb H37Rv acute and chronic infection mouse model to further evaluate its antituberculosis effect in vivo.The results showed that compound 5a demonstrated more significant efficacy in reducing the bacterial burden in lungs at doses of 300 mg/kg(P<0.0001)and 100 mg/kg(P<0.001)compared to the untreated group in the Mtb H37Rv acute infection mouse model.Additionally,the combination of ALZE(A,amikacin;L,levofloxacin;Z,pyrazinamide;E,ethambutal)plus 5a group effectively lowered the bacterial burden in lungs(P<0.05)compared with ALZE group.On the other hand,compound 5a significantly lowered the bacterial burden in lungs even at a dose of 30 mg/kg(P<0.05)compared with the untreated group in the Mtb H37Rv chronic infection mouse model And 5a demonstrated more significant efficacy lowered the bacterial burden in lungs at higher dose i.e.300 mg/kg(P<0.001)100 mg/kg(P<0.001).Additionally,the combination of RH(R,rifampin;H,isoniazide)plus 5a group significantly lowered the bacterial burden in lungs(P<0.01)compared with RH group.Furthermore,the combination of LZE(L,levofloxacin;Z,pyrazinamide;E,ethambutal)plus 5a group also significantly lowered the bacterial burden in lungs(P<0.01)compared with LZE group.To establish the mutation associated with 5a drug resistance,gene amplification and sequencing analysis was performed on the selected compound 5a drug-resistant mutant strains.The sequencing results showed that the katG gene mutation of Mtb is associated with resistance to 5a.This study unearthed two compounds with strong antituberculosis activity both in vitro and in vivo.Although compound 16j has significant antituberculosis activity in vivo.It has partial synergistic effect when combined with SMX in vitro,and DHFR might be its potential target.On the other hand,compound 5a has significant antituberculosis activity when used alone in vivo.It has strong effect when used in combination with either first-line or second-line antituberculosis drugs in the Mtb H37Rv chronic infected mouse model.Notably,the mutation of katG gene in Mtb leads to its resistance suggesting that the NADH-dependent enoyl-[ACP]reductase(InhA)might be its potential target.