Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

Abstract

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target

“…Since anziaic acid was reported as a Topo I poison, it was tested for MIC against both wildtype M. tuberculosis and topA/OE strains. We hoped that the topA/ OE cells would display increased sensitivity to anziaic acid as is the case with camptothecin in human Topo I overexpressing cells [44] and imipramine, norclomipramine in case of mycobacteria [51]. However, MIC of anziaic acid was the same (Table 2) both in wildtype and topA/OE strains suggesting a different mechanism of action.…”

Learning from the past for TB drug discovery in the future

“…Identify compounds likely to be metabolically activated (e.g., norclomipramine is a metabolite of clomipramine, and only the former is active vs M. tuberculosis topoisomerase I in vitro [89]).…”

“…The identification of the proton pump inhibitor lansoprazole which is reduced to an active metabolite intracellularly and shown to possess in vivo activity in mouse [87] is a recent example derived from screening FDA-approved drugs in vitro . Target-based computational models have led to approved drug compounds that were not previously considered as antibiotics, with good activity against M. tuberculosis Topo I (e.g., amsacrine, imipramine and norclomipramine) [88,89] and ThyX (idebenone) [90] but with poorer MICs. So this gets to the additional challenge of finding molecules that hit the sweet spot of in vitro activity at the target and in the whole cell, and that is before trying in vivo .…”

Targeting Bacterial Topoisomerases: How to Counter Mechanisms of Resistance

“…Several small molecules have been reported recently as inhibitors of M. tuberculosis topoisomerase I in enzyme assays, with further biological evidence to support topoisomerase I as the cellular target for antimycobacterial growth inhibition [91,94]. The MIC for growth inhibition is shifted in different directions when M. tuberculosis topoisomerase I is overexpressed, depending on the mechanism of inhibition.…”