An essential type I nitroreductase from Leishmania major can be used to activate leishmanicidal prodrugs.
Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR(+/-) heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR(+/-) heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.
From the Queen Mary Pre-Clinical Drug Discovery Group, School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom.
Andrew A
AA
Voak
Vithurshaa
V
Gobalakrishnapillai
Karin
K
Seifert
Edina
E
Balczo
Longqin
L
Hu
Belinda S
BS
Hall
Shane R
SR
Wilkinson
D000483:Alleles; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D005486:Flavin Mononucleotide / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D007700:Kinetics; D018320:Leishmania major / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D051379:Mice; D008807:Mice, Inbred BALB C; D009150:Mustard Compounds / Q000737:chemistry / Q000494:pharmacology; D009593:Nitroimidazoles / Q000737:chemistry / Q000494:pharmacology; D009601:Nitroreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity / Q000187:drug effects; D014344:Trypanocidal Agents / Q000737:chemistry / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D009150:Mustard Compounds; D009593:Nitroimidazoles; D011355:Prodrugs; D014344:Trypanocidal Agents; D005486:Flavin Mononucleotide; D009601:Nitroreductases; C009999:benzonidazole
Drug Discovery; Drug Screening; Oxidation-Reduction; Parasite; Trypanosoma brucei; Trypanosome