Optimization of pyrrolamides as mycobacterial GyrB ATPase inhibitors: structure-activity relationship and in vivo efficacy in a mouse model of tuberculosis.

Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 µg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 µg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.

Essentiality and functional analysis of type I and type III pantothenate kinases of Mycobacterium tuberculosis.

Pantothenate kinase, an essential enzyme in bacteria and eukaryotes, is involved in catalysing the first step of conversion of pantothenate to coenzyme A (CoA). Three isoforms (type I, II and III) of this enzyme have been reported from various organisms, which can be differentiated from each other on the basis of their biochemical and structural characteristics. Though most bacteria carry only one of the isoforms of pantothenate kinases, some of them possess two isoforms. The physiological relevance of the presence of two types of isozymes in a single organism is not clear. Mycobacterium tuberculosis, an intracellular pathogen, possesses two isoforms of pantothenate kinases (CoaA and CoaX) belonging to type I and III. In order to determine which pantothenate kinase is essential in mycobacteria, we performed gene inactivation of coaA and coaX of M. tuberculosis individually. It was found that coaA could only be inactivated in the presence of an extra copy of the gene, while coaX could be inactivated in the wild-type cells, proving that CoaA is the essential pantothenate kinase in M. tuberculosis. Additionally, the coaA gene of M. tuberculosis was able to complement a temperature-sensitive coaA mutant of Escherichia coli at a non-permissive temperature while coaX could not. The coaX deletion mutant showed no growth defects in vitro, in macrophages or in mice. Taken together, our data suggest that CoaX, which is essential in Bacillus anthracis and thus had been suggested to be a drug target in this organism, might not be a valid target in M. tuberculosis. We have established that the type I isoform, CoaA, is the essential pantothenate kinase in M. tuberculosis and thus can be explored as a drug target.