RESUMO
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.
Assuntos
Leishmania major/enzimologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Tripanossomicidas/farmacologia , Alelos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Mononucleotídeo de Flavina/metabolismo , Heterozigoto , Humanos , Cinética , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Mostarda/química , Compostos de Mostarda/farmacologia , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Nitrorredutases/antagonistas & inibidores , Pró-Fármacos/química , Especificidade por Substrato/efeitos dos fármacos , Tripanossomicidas/químicaRESUMO
A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.