Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence have revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e. CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent utilized in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5 - its highly homologous counterpart - is susceptible to inactivation by BBR. Using three structurally-distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities towards MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation where apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing pre-incubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, non-covalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared to CYP3A4 which further substantiated our experimental observations. Significance Statement While we have previously demonstrated that BBR inactivates CYP3A4, it remains uninterrogated if it could also elicit MBI in CYP3A5, which possesses considerable sequence homology. Here, we report that BBR exhibits differential irreversible and reversible interactions with CYP3A4 and CYP3A5 and provided potential mechanistic insights on the structural molecular determinants underpinning their diverging interaction profiles. Our findings reinforce the importance of discerning between the kinetic behavior of CYP3A due to their propensities for distinct interaction profiles with a common substrate.