Reduction of Nilutamide by NO Synthases:  Implications for the Adverse Effects of This Nitroaromatic Antiandrogen Drug

Abstract

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of l-arginine to l-citrulline with formation of the widespread signal molecule NO. Beside their fundamental role in NO biosynthesis, these enzymes are also involved in the formation of reactive oxygen species and in the interactions with some xenobiotic compounds. Nilutamide is a nonsteroidal antiandrogen that behaves as a competitive antagonist of the androgen receptors and is proposed in the treatment of metastatic prostatic carcinoma. However, therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions mediated by toxic mechanism(s), which remain(s) poorly investigated. Here, we studied the interaction of NOSs with nilutamide. Our results show that the purified recombinant neuronal NOS reduced the nitroaromatic nilutamide to the corresponding hydroxylamine. The reduction of nilutamide catalyzed by neuronal NOS proceeded with intermediate formation of a nitro anion free radical easily observed by EPR, was insensitive to the addition of the usual heme ligands and l-arginine analogues, but strongly inhibited by O(2) and a flavin/NADPH binding inhibitor. Involvement of the reductase domain of nNOS in the reduction of nilutamide was confirmed by (i) the ability of the isolated reductase domain of nNOS to catalyze the reaction and (ii) the stimulating effect of Ca(2+)/calmodulin on the accumulation of hydroxylamine and nitro anion radical. In a similar manner, the recombinant inducible and endothelial NOS isoforms also displayed nitroreductase activity, albeit with lower yields. The selective reduction of nilutamide to its hydroxylamino derivative by the NOSs could explain some of the toxic effects of this drug.

Bioactivation and Hepatotoxicity of Nitroaromatic Drugs

“…For example, it has been shown that recombinant neuronal NOS reduced nilutamide to the corresponding hydroxylamine via the formation of a nitroanion radical. This reaction was inhibited by molecular oxygen and a flavin/ NADPH binding inhibitor and stimulated by Ca 2+ /calmodulin, providing strong evidence that NOS was involved [26,28]. One of the underlying reasons for this non-classical reaction of NOS is that NOS has a reductase domain, which shares close homology with CYP reductase [29].…”

“…Recently NOS has been implicated in the nitroreduction of nitroarenes [26]. NOSes are flavohemoproteins that catalyze the oxidation of L-arginine to L-citrulline resulting in the formation of nitric oxide.…”

“…Rare cases of pneumotoxicity and hepatotoxicity have been described [23,26]. Nilutamide is a nitroaromatic drug that is p-substituted and o-substituted by a trifluoromethyl moiety (Fig.…”

“…cals are not very reactive towards biological nucleophiles [8]. The new concept involving NOS can, however, provide an explanation for the greater reactivity of nilutamide and other nitroarenes towards biological nucleophiles via NOScatalyzed subsequent production of hydroxylamines [26]. For example, the hydroxylamine can be further oxidized to the nitroso (which is an electrophile) or undergo acetylation to the N-acetoxy derivative, forming a highly reactive nitrenium ion that alkylates proteins or DNA [96].…”

FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells

“…However, the one-electron reductases in human tumour cells are poorly characterized. The diflavin reductase POR (NADPH:cytochrome P450 oxidoreductase) is considered to be a major contributor [16,19], but other flavoproteins have been shown to be capable of catalysing one-electron reduction of nitro compounds, including MTRR (methionine synthase reductase), NDOR1 (NADPH-dependent diflavin oxidoreductase 1) [16], NOS (nitric oxide synthase) isoforms NOS1, NOS2A and NOS2B [20,21], thioredoxin reductase [22], CYB5R3 (NADHdependent cytochrome b 5 reductase 3) [23], aldehyde oxidase [24,25] and xanthine oxidase [26,27]. However, there is little information on the contribution that these or other enzymes make, at physiological levels of expression, to bioreductive prodrug activation in tumours.…”