ParA: A Novel Target for Anti-Tubercular Drug Discovery

Abstract

<p>Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme.</p>