The Curious Case of Benzbromarone: Insight into Super-Inhibition of Cytochrome P450

Abhinav Parashar,Mahesh Potnuru,Sudeep Kumar Gade,Nandita Madhavan,Kelath Murali Manoj,Daniel S Sem

doi:10.1371/journal.pone.0089967

Abstract

Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing “super-inhibition” phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

Highlights

Cytochrome P450 (CYP) family of enzymes mediate the metabolism of a majority of xenobiotics in mammals
It was found that while the crystallized chimera/mutant of CYP2C9 (1OG5) had no positively charged residues within the active site, Arg108 was a probable candidate for binding of anionic moieties in the active site of CYP2C9 (1R9O) [1,3]
It was argued that the 1R9O structure better explained the catalytic activities of CYP2C9

Summary

Introduction

Cytochrome P450 (CYP) family of enzymes mediate the metabolism of a majority of xenobiotics in mammals. Among the inhibitors reported for CYPs, benzbromarone (BzBr) and its iodinated analogue, benziodarone (BzIr) have been suggested to be the most potent for CYP2C9, with Ki value of #1 nM [4,5,6]. One of us found that the values of inhibition constants derived from experimental rate measurements ‘stretched’ the intuitive understanding afforded by the theoretical model. This is because the practical enzyme inhibition assays are performed with tens to hundreds of nM CYPs and ,100 mM concentration of substrates (at ,10X KM). The work provides an alternative explanation for the inhibitions observed and gives key insights into the mechanistic aspects of cytochrome P450 mediated redox metabolism

Methods

Results

Conclusion