Abstract
Mounting evidence has 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 used 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 toward 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 wherein apparent Vmax increased and Km 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 preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations.
SIGNIFICANCE STATEMENT Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.
Footnotes
- Received February 4, 2021.
- Accepted June 21, 2021.
↵1 L.W.T.T. and R.K.V contributed equally to the work.
This work is supported by the Agency for Science, Technology and Research (A*STAR) Industry Alignment Fund–Pre-Positioning (IAF-PP) Funding [Grant H18/01/a0/C14] provided to H.F and E.C.Y.C and the National University Heart Centre Singapore (NUHCS) Cardiovascular Research Institute (CVRI)–Core Fund [Grant NUHSRO/2019/082/Core] and SCEPTRE CG Seed Grant [Grant NMRC/CG/M008/2017] provided to E.C.Y.C. L.W.T.T is supported by the National University of Singapore (NUS) President’s Graduate Fellowship (PGF).
The authors declare that they have no conflicts of interest with the contents of this article.
↵
This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|