The
nitroimidazole fexinidazole has potential as a safe and effective oral
drug therapy for the treatment of
visceral leishmaniasis. To date, nitroheterocyclics have not been used in the treatment of
leishmaniasis, and relatively little is known about their mechanism of action. In African trypanosomes, nitro drugs are reductively activated by a type I
nitroreductase (NTR), absent in mammalian cells. Modulation of
nitroreductase levels in Trypanosoma brucei directly affected sensitivity to
nitro compounds, with reduced concentrations of the
enzyme leading to moderate nitro drug resistance. In view of the progression of
fexinidazole into clinical development for
visceral leishmaniasis, here we assess the essentiality of the
nitroreductase in Leishmania donovani and the effect of modulating
nitroreductase levels on susceptibility to
fexinidazole. The failure to directly replace both endogenous copies of the NTR gene, except in the presence of an ectopic copy of the gene, suggests that the NTR gene is essential for the growth and survival of L. donovani promastigotes. Loss of a single chromosomal copy of the L. donovani NTR gene resulted in parasites that were mildly resistant (<2-fold) to the predominant in vivo metabolite of
fexinidazole, while parasites overexpressing NTR were 18-fold more susceptible. These data confirm that Leishmania NTR plays a pivotal role in
fexinidazole activation. Reliance on a single
enzyme for
prodrug activation may leave
fexinidazole vulnerable to the emergence of drug resistance. However, the essentiality of the NTR in L. donovani promastigotes, combined with the limited resistance shown by NTR single knockout cells, suggests that the potential for the spread of NTR-based resistance to
fexinidazole may be limited.