Benzodiazepine class drugs act as central nervous system depressants and are among the most commonly prescribed medications in the Western hemisphere. Clonazepam (Klonopin), used to treat seizures and panic disorders, has important therapeutic applications. Flunitrazepam (Rohypnol), in contrast, has a high abuse potential: it is often used as a ‘date-rape’ drug due to its sedative and amnesia-inducing properties. Aldoketoreductases (AKRs) are a family of enzymes with 15 members in humans. The AKR1C class is important in steroid hormone metabolism; however, they have also been implicated in drug metabolism. Notably, flunitrazepam is metabolized by AKR1C3, although clonazepam is not metabolized by this enzyme in spite of its close structural similarity. Flunitrazepam is not metabolized by any of the other three members of the AKR1C, or hydroxysteroid dehydrogenase, class of enzymes: AKR1C1, AKR1C2, and AKR1C4. The aims of this project are to understand i) the specific interactions between flunitrazepam and AKR1C3 that enable it to be metabolized by this enzyme and not the other AKR1C enzymes, and ii) why clonazepam is not metabolized by AKR1C3. This was done using molecular modeling and simulation tools including automated protein ligand docking and molecular dynamics simulations. 

Molecular dynamics simulations revealed that flunitrazepam rapidly approaches the AKR1C3 binding site and consistently remains within hydrogen bonding distance of the hydride donor on NADPH, with the nitro group staying within 4 Å of NADPH 95% of the time. On the other hand, the nitro group on clonazepam is within 4 Å of NADPH only 6.8% of the time, indicating a lack of significant interaction. The molecular dynamics simulations help explain why flunitrazepam is a substrate of AKR1C3 and clonazepam is not. Analysis of the residues near the methyl group of flunitrazepam and the nitrogen proton on clonazepam suggests that the interaction of MET120 with the flunitrazepam methyl group plays a key role in positioning the drug close enough to the catalytic center. Furthermore, in AKR1C1 and AKR1C, residue 120 is replaced by the smaller, less hydrophobic residue valine, which would explain why  flunitrazepam cannot be metabolized by these two AKR forms. Docking results indicated that flunitrazepam does not orient its nitro group towards NADPH in the AKR1C1, AKR1C2, or AKR1C4 binding sites, corroborating the AKR1C3 substrate specificity. These findings enhance our understanding of benzodiazepine metabolism, significant in regards to flunitrazepam’s high potential for abuse.