Cytochrome P450's (CYPs) are a major source of metabolic drug interactions. The allosteric behavior of CYPs, including the major drug metabolizing isoform CYP3A4, continues to confound in vitro - In vivo correlations and the prediction of drug interactions based on in vitro data. The molecular mechanisms that confer allosteric behavior are incompletely understood, and improved mechanistic models are likely to improve predictive drug metabolism. Moreover, although allosteric behavior in vitro is well known and widely observed, examples of in vivo allosterism are limited. Among the multiple mechanisms that contribute, occupancy by small molecule drugs at a peripheral effector site of CYP3A4, and conformational changes induced by the electron transfer partner Cytochrome bs (Cyt bs), are likely to converge on a set of common effects, wherein the active site is more efficiently desolvated or 'well-packed'through protein-protein interactions or multiple drug binding. In turn this, hypothetically, leads to more efficient coupling of NADPH consumption and O2 reduction, concomitant with drug oxidation, with decreased 'uncoupling'to form reduced oxygen species. This proposal aims to: 1) increase our understanding of allosteric CYP mechanisms with model probe drugs, and 2) to complete a comparative, and mechanistic, analysis of CYP-Cyt b5 interactions. The fluorescent probes Nile Red and TNS provide a comparison of the active site hydration and steric constraints of CYP3A4 when the peripheral allosteric site is occupied or empty. Mass spectrometry and computational models provide methods to map binding interactions in Cyt b5-CYP complexes and the resulting conformational changes linked to effector functions of Cyt bs. Each of these approaches will be combined with functional studies to characterize reaction intermediates that determine the efficiency of the flux through the catalytic cycle. A major goal of these studies is to develop their translational utility in conceptually analogous studies with P-glycoprotein in Project 4 of this Program. In addition, the importance of in vivo allosteric effects remains speculative, and the final aim, aim 3, of this proposal will utilize a novel in vivo, human, clinical study to explore heterotropic effects between the antiepileptic drug carbamazapine and the antianxiety drug midazolam. This study not only seeks to establish definitive proof-of-principle for in vivo allosteric effects in human CYPs, but also to provide a platform experimental design to be exploited in other in vivo studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM032165-28
Application #
8118441
Study Section
Special Emphasis Panel (ZGM1)
Project Start
Project End
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
28
Fiscal Year
2010
Total Cost
$251,654
Indirect Cost
Name
University of Washington
Department
Type
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Wong, Timothy; Wang, Zhican; Chapron, Brian D et al. (2018) Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans. Drug Metab Dispos 46:367-379
Shirasaka, Y; Chaudhry, A S; McDonald, M et al. (2016) Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content. Pharmacogenomics J 16:375-87
Manoj, Kelath Murali; Parashar, Abhinav; Gade, Sudeep K et al. (2016) Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes. Front Pharmacol 7:161
Stamper, Brendan D; Garcia, Michael L; Nguyen, Duy Q et al. (2015) p53 Contributes to Differentiating Gene Expression Following Exposure to Acetaminophen and Its Less Hepatotoxic Regioisomer Both In Vitro and In Vivo. Gene Regul Syst Bio 9:1-14
McDonald, Matthew G; Au, Nicholas T; Rettie, Allan E (2015) P450-Based Drug-Drug Interactions of Amiodarone and its Metabolites: Diversity of Inhibitory Mechanisms. Drug Metab Dispos 43:1661-9
Chaudhry, Amarjit S; Prasad, Bhagwat; Shirasaka, Yoshiyuki et al. (2015) The CYP2C19 Intron 2 Branch Point SNP is the Ancestral Polymorphism Contributing to the Poor Metabolizer Phenotype in Livers with CYP2C19*35 and CYP2C19*2 Alleles. Drug Metab Dispos 43:1226-35
Liu, Li; Collier, Ann C; Link, Jeanne M et al. (2015) Modulation of P-glycoprotein at the Human Blood-Brain Barrier by Quinidine or Rifampin Treatment: A Positron Emission Tomography Imaging Study. Drug Metab Dispos 43:1795-804
Ho, Han Kiat; Chan, James Chun Yip; Hardy, Klarissa D et al. (2015) Mechanism-based inactivation of CYP450 enzymes: a case study of lapatinib. Drug Metab Rev 47:21-8
Chapron, Brian; Risler, Linda; Phillips, Brian et al. (2015) Reversible, time-dependent inhibition of CYP3A-mediated metabolism of midazolam and tacrolimus by telaprevir in human liver microsomes. J Pharm Pharm Sci 18:101-11
Caudle, K E; Rettie, A E; Whirl-Carrillo, M et al. (2014) Clinical pharmacogenetics implementation consortium guidelines for CYP2C9 and HLA-B genotypes and phenytoin dosing. Clin Pharmacol Ther 96:542-8

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