Evaluating and exploiting the function of a Leishmania major type I nitroreductase in the development of novel leishmanicidal prodrugs.

Abstract

Leishmania are protozoan parasites responsible for the range of infections collectively known as leishmaniasis. Currently, drugs represent the only course of treatment. The nitroaromatic prodrugs are a class of agents that are used clinically used to treat trypanosomatid diseases; and in the parasites Trypanosoma brucei and Trypanosoma cruzi these compounds undergo reduction activation by enzymes homologous with bacterial type I nitroreductases (NTRs). From the Leishmania major genome database, we have identified a protein (LmNTR) that could catalyse this reaction. Based on co-factor, oxygen-insensitive activity and substrate range, we demonstrate that the LmNTR displays many characteristics of its bacterial counterparts. Gene deletion studies revealed that LmNTR is essential to the insect and mammalian stages of L. major. Null mutant parasites could not be generated while loss of a single LmNTR allele in the insect form conferred resistance to a range of nitroaromatic compounds without affecting their growth rate or ability to differentiate into infectious forms. Heterozygous lines could not establish an infection in vitro using a tissue culture model, or in vivo, in mice. As NTRs are absent from most eukaryotes, with trypanosomatids being a major exception, we exploit this difference to evaluate a library of nitroaromatic compounds against L. major parasites. Biochemical screens against the purified enzyme revealed that several compounds were effective substrates for LmNTR, generating higher activities than nifurtimox, the clinically used nitro-based agent that targets trypanosomes. Using phenotypic screens, we demonstrated that growth inhibition mirrored enzyme activity, with the most potent compounds generating IC50’s <100 nM whilst having little effect on mammalian cells. L. major NTR was shown to play a key role in parasite killing as heterozygous lines displayed resistance to the compounds. In conclusion, we have shown that LmNTR is essential to the parasite and by exploiting its prodrug activating properties, identified several novel agents that provide new lead structures to treat leishmanial infections