Drug discovery pipelines for the "
neglected diseases" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic
nitro-compounds (R)-PA-824, DNDI-VL-2098 and
delamanid have been identified as potential candidates for the treatment of
visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative
NAD(P)H oxidase as the activating
nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single
cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated
protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic
nitro-compounds, but only marginally to the monocyclic nitro-drugs,
nifurtimox and
fexinidazole sulfone, known to be activated by a mitochondrial
oxygen-insensitive
nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic
nitro-compounds and only marginally resistant to
nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic
nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.