Date Published: November 3, 2016
Publisher: Public Library of Science
Author(s): Susan Wyllie, Adam J. Roberts, Suzanne Norval, Stephen Patterson, Bernardo J. Foth, Matthew Berriman, Kevin D. Read, Alan H. Fairlamb, Stephen M. Beverley.
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.
New, safer and more effective treatments are required for visceral leishmaniasis (VL), a disease endemic in parts of Asia, Africa and South America. VL results from infection with the protozoan parasites Leishmania donovani or L. infantum and is responsible for ~50,000 deaths per annum, with the number of cases estimated between 200,000 and 400,000 . In 95% of cases, death can be prevented by timely and appropriate drug therapy ; however, current treatment options are far from ideal . At present, miltefosine and liposomal amphotericin B are considered the front-line therapies and, while both drugs are considerably more effective than previous treatment options, they have their limitations. The principal drawbacks of amphotericin B include high treatment costs, the requirement of a cold chain for distribution and storage, an intravenous route of administration and unresponsiveness in some Sudanese VL patients . Problems associated with miltefosine, the only oral drug, are its teratogenicity and high potential to develop resistance . Thus, there is a pressing need for better, safer efficacious drugs that are fit-for-purpose in resource-poor settings.
Drug discovery pipelines for the “neglected diseases” are now heavily populated with nitroheterocyclic compounds. Following the success of nifurtimox as part of NECT  and the re-discovery of fexinidazole [7,8,20], researchers have been quick to recognise and exploit the therapeutic potential of these compound classes. However, the development of multiple compounds with a likely shared mode of action for any disease indication is not without significant risk. An over-emphasis on one compound class can leave drug pipelines vulnerable to multiple compound failures associated with a single resistance mechanism. Specifically, it is well established that parasites resistant to one nitro-drug are often cross-resistant to a second ; for example nifurtimox-resistant T. brucei are cross-resistant to fexinidazole and vice versa [11,16]. Here, cross-resistance has been largely explained by a common, nitroreductase-related mechanism of drug activation [13,21]. This has made researchers wary of developing further nitro-compounds for the treatment of the trypanosomatid diseases. In this study we have confirmed that several bicyclic nitro-drugs, either in preclinical development or demonstrating promising anti-leishmanial activity, are not activated via NTR1, known to activate monocyclic nitro-compounds nifurtimox, benznidazole and fexinidazole [7,10,12,22]. Importantly, resistance to bicyclic nitro-compounds in Leishmania promastigotes does not result in striking levels of cross-resistance to NTR1-activated compounds. 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.