ABSTRACT
Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Mycobacterium tuberculosis/drug effects , Pyrazoles/pharmacology , Spiro Compounds/pharmacology , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/therapeutic use , Bacterial Proteins/metabolism , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Chromatography, Thin Layer , Cord Factors , Disease Models, Animal , Dogs , Drug Resistance, Bacterial , Genotype , Hep G2 Cells , Humans , Kinetics , Mice , Microbial Sensitivity Tests , Microbial Viability/drug effects , Mutation/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/therapeutic use , Rats , Spiro Compounds/chemistry , Spiro Compounds/pharmacokinetics , Spiro Compounds/therapeutic use , Treatment Outcome , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1-4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism (937)ACC>(937)GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.