title: 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.
authors.affiliation: School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Parashar
authors.affiliation: School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.
authors.forename: Sudeep Kumar
authors.initials: SK
authors.lastname: Gade
authors.affiliation: Department of Chemistry, IIT Madras, Chennai, Tamil Nadu, India.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Potnuru
authors.affiliation: Department of Chemistry, IIT Madras, Chennai, Tamil Nadu, India.
authors.forename: Nandita
authors.initials: N
authors.lastname: Madhavan
authors.affiliation: REDOx Lab, Biotechnology and Healthcare Division, PSG Institute of Advanced Studies, Peelamedu, Tamil Nadu, India.
authors.forename: Kelath Murali
authors.initials: KM
authors.lastname: Manoj
mesh_terms: D001553:Benzbromarone / Q000494:pharmacology*; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004008:Diclofenac / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004008:Diclofenac; D001553:Benzbromarone; D003577:Cytochrome P-450 Enzyme System