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

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.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions

“…From our studies, it is evident that P450 inhibition profiles could also be difficult to predict/interpret, with the erstwhile hypothesis as foundation. In this study, we have shown that both one electron and two electron reactions occurring in the aqueous phase is modulated by additives and therefore, it is only natural that the same effect be expected or amplified in the phospholipid interfaces where P450s catalyze reactions, This was in fact corroborated by our recent works [16,18,21]. Heme enzymes (and perhaps, other such redox enzymes) are unlike their classical counterparts in that they work via a "murburn" ('mured burning' or mediate unrestricted burning) mechanism [58] and not necessarily by 'induced fit' or 'lock & key' hypotheses.…”

“…Several other modulations and competitive inhibitions are also reported in heme-enzyme systems [49,50]. However, we had expanded the paradigm of redox enzyme "activations" when we reported the intriguing enhancement of reactions by low concentrations of known Type I & II hemoprotein binding "inhibitors" [14,21,22] and redox active molecules [15]. The current manuscript establishes the idea that trace amounts of additive molecules can also inhibit heme-enzyme mediated one-electron oxidations.…”

“…(We had shown earlier that K i was lower for lower inhibitor concentrations, in a case of P450 inhibition [21]. This is also a fundamental contradiction to the application of inhibitor-enzyme binding paradigm for heme enzymes.)…”

Murburn concept explains the acutely lethal effect of cyanide

“…In the various hemeperoxiases and mixed oxidase cytochrome P450 systems, similar prevailing fallacies were pointed out and the kinetic/mechanistic phenomena were explained with murburn concept. 3,4,12,13,20,21…”

“…[1][2] Recently, an oxygen-centric mechanism called murburn (from 'mured burning') concept was proposed to explain the physiology of xenobiotic metabolism and cellular respiration. [3][4][5][6][7] The new proposal states that diffusible reactive oxygen species (DROS) generated at the mitochondrial complexes directly serve as the coupling agents of oxidative phosphorylation. It was proposed that cyanide ion-radical mediated catalysis, and not cyanide binding to heme (as currently held), is the primary rationale for cyanide lethality.…”