Professor and Chairman, Medicinal Chemistry
PH: 206 543 2224
Metabolism by the cytochrome P450s is the principal means whereby lipid-soluble drugs and compounds foreign to the body are converted to water-soluble derivatives that can be readily excreted. This is a beneficial effect of the enzyme system. However, it is also well recognized that P450-mediated bioactivation of drugs and other xenobiotics is an important mechanism of chemical toxicity. Moreover, unexpected interruptions in P450 activity, due to genetic variation or administration of agents that inhibit P450 activity, can cause serious adverse drug reactions and contribute to disease states.
Research in our laboratory focuses primarily on the biochemistry and pharmacogenetics of the human CYP2 and CYP4 families of P450s. Human CYP2C9, for example, is the primary catalyst of (S)-warfarin metabolism. This anticoagulant drug is very difficult to dose correctly, and there are many drug-drug and drug-gene interactions associated with its use. An important goal for the laboratory is to define sources of inter-individual variability in warfarin dosing which can span a 100-fold range. Recently, we found that CYP4F2 is a vitamin K oxidase and polymorphisms in this gene affect warfarin dose likely by modulating hepatic vitamin K concentrations. Drug-drug interactions that involve CYP2C9 are pharmacokinetic in origin. Currently, we are focusing attention on new pharmacodynamic target genes in the blood clotting pathway, such as VKORC1, which encodes the warfarin receptor.
CYP4B1 metabolizes a host of pro-toxins, including furans, aromatic amines and certain fatty acids to reactive intermediates that can damage the cell. In this regard, 4B1 is a curious member of the CYP4 family because generally these enzymes have a restricted substrate specificity that does not extend much beyond endogenous fatty acids, such as arachidonate. CYP4V2 is a novel ‘orphan P450’ whose substrate specificity is unknown. Intriguingly, polymorphisms in CYP4V2 are found in patients suffering from the eye disease, Bietti’s Crystalline Dystrophy (BCD). We are attempting to ‘de-orphanize’ CYP4V2 and, in the process, discover which homeostatic mechanisms have been disrupted in BCD patients.
In this work we use protein engineering coupled with conventional protein biochemistry methods for the expression and isolation of proteins and mutants of interest from heterologous hosts such as E.coli and insect cells. We also make extensive use of mass spectrometry (MS) for analyte quantification, including evaluation of structural changes in mutant proteins and lipidomic analysis to probe changes in endogenous metabolism due to CYP4V2 polymorphisms. We are also developing CYP4 knockout mouse models to study mechanisms of CYP4V2 and CYP4B1 toxicity in vivo. Synthetic chemistry comes into play in the preparation of new substrates, inhibitors and metabolites for P450s of interest. Our long-term goals are to understand how structure and function are related for these important P450 enzymes.
RECENT SELECTED PUBLICATIONS
- Danese E, Montagnana M, Johnson JA, Rettie AE, Zambon CF, et al. "Impact of the CYP4F2 p.V433M polymorphism on coumarin dose requirement: systematic review and meta-analysis." Clin Pharmacol Ther. 92(6):746-56 (Dec 2012).
- MacDonald MG, Au N, Wittkowsky AK, and Rettie AE. "Warfarin-Amiodarone Drug-Drug Interactions: Determination of [I]u/Ki,u for Amiodarone and its Plasma Metabolites Suggests a Major Role for a Minor Metabolite." Clin Pharmacol Ther., 91(4):709-17 (Apr 2012).
- Parkinson OT, Kelly EJ, Whittington D, and Rettie AE. "Bioactivation of 4-Ipomeanol by a CYP4 Enzyme from Cow Lung and Inhibition by HET0016." J Vet Pharmacol Ther., doi: 10.1111/j.1365-2885.2011.01339.x (15 Sep 2011).
- Innocenti F, Cooper GM, Stanaway IB, Gamazon ER, Smith JD, Mirkov S, Ramirez J, Liu W, Lin YS, Moloney C, Aldred SF, Trinklein ND, Schuetz E, Nickerson DA, Thummel KE, Rieder MJ, Rettie AE, Ratain MJ, Cox NJ, and Brown CD. "Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue." PloS Genetics, 7:(5) e1002078 (2011).
- Kelly EJ, Nakano M, Rohatgi P, Yarov-Yarovoy, and Rettie AE. "Finding Homes for Orphan Cytochrome P450s: CYP4V2 and CYP4F22 in Disease States." Molecular Interventions, 11:124-132 (2011).
- Baillie TA, Rettie AE. "Role of Biotransformation in Drug-Induced Toxicity: Influence of Intra- and Inter-Species Differences in Drug Metabolism." Drug Metab Pharmacokinet., 26:15-29 (2011).
- Roberts AG, Cheesman MJ, Primak A, Bowman MK, Atkins WM, Rettie AE. "Intramolecular Heme Ligation of the Cytochrome P450 2C9 R108H Mutant Demonstrates Pronounced Conformational Flexibility of the B-C Loop Region: Implications for Substrate Binding." Biochemistry. 2010 September 21 [Epub ahead of print].
- Nakano M, Kelly EJ and Rettie AE. "Expression and characterization of CYP4V2 as a fatty acid omega-hydroxylase." Drug Metab Dispos. 37:2119-22 (2009).
- McDonald MG, Rieder MJ, Nakano M, Hsia CK and Rettie AE. "CYP4F2 is a vitamin K1 oxidase: An explanation for altered warfarin dose in carriers of the V433M variant." Mol Pharmacol. 75:1337-46 (2009).
- Mosher CM, Tai G and Rettie AE. "CYP2C9 amino acid residues influencing phenytoin turnover and metabolite regio- and stereochemistry." J Pharmacol Exp Ther. 329:938-44 (2009).
- Mosher CM, Hummel MA, Tracy TS and Rettie AE. "Functional analysis of phenylalanine residues in the active site of CYP2C9." Biochemistry 47:11725-34 (2008).
- Cooper GM, Johnson JA, Langaee TY, Feng H, Stanaway IB, Schwartz U Ritchie MD, Stein M, Roden DM, Smith JD, Veenstra DL, Rettie AE, and Rieder MJ. "A Whole-Genome Scan for Common Genetic Variants with a Large Influence on Warfarin Maintenance Dose." Blood 112:1022-1027 (2008).