Metabolic activation of flunitrazepam via nitroreduction mediated by aldo-keto reductase 1C3

Aldo-keto reductases (AKRs) are a superfamily of NAD(P)(H)-dependent oxidoreductases (phase I enzymes) that reduce aldehydes and ketones to primary and secondary alcohols, respectively. Four percent of xenobiotic biotransformation has been attributed to AKRs, although this is likely an underestimate because this is based on their ability to act as carbonyl reductases. AKRs also have an emerging role in nitroreduction. We screened 6 nitro-containing drugs (flunitrazepam, clonazepam, nimesulide, nilutamide, flutamide, and chloramphenicol) as substrates against recombinant human AKR1C1, AKR1C2, AKR1C3, and AKR1C4, using discontinuous enzymatic assays. AKR1C3 was able to reduce flunitrazepam to 7-amino-flunitrazepam. Metabolites formed during the reaction were identified by tandem mass spectrometry, which confirmed the formation of nitroso and hydroxylamino intermediates. The role of AKR1C3 was confirmed in HepG2 cells since AKR1C3-specific inhibitors blocked the formation of 7-amino-flunitrazepam. Interestingly, clonazepam (which lacks the tertiary amide of flunitrazepam) was not metabolized by AKR1C3. Molecular docking and dynamics simulations indicated that residue 120 (methionine) in AKR1C3 plays an important role in flunitrazepam-protein binding. Our computational approach suggested that the interaction between the flunitrazepam and AKR1C1, AKR1C2, and AKR1C4 is unlikely to occur and supported our experimental results. Our findings show that AKR1C3 can reduce flunitrazepam to reactive nitroso intermediates that could enhance drug toxicity and that flunitrazepam inhibits AKR1C2, which is responsible for the formation of the neuroactive steroid allopregnanolone. We conclude that both AKR1C3 and AKR1C2 may have an important role in flunitrazepam drug response. SIGNIFICANCE STATEMENT: Aldo-keto reductase (AKR)1C3 reduces flunitrazepam to 7-amino-flunitrazepam through the formation of reactive nitroso and hydroxylamino intermediates, and by inhibiting AKR1C2, flunitrazepam may reduce the formation of the neuroactive steroid allopregnanolone.