Metabolism by the cytochrome P450's 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. However, unexpected interruptions in P450 activity, due to genetic variation or administration of drugs that inhibit P450 activity, can cause serious adverse drug reactions. Moreover, P450-mediated bioactivation of drugs and xenobiotics is a well recognized mechanism of chemical toxicity.
Research in our laboratory focuses on the biochemistry and chemistry associated with metabolic transformations dependent on the human CYP2C (2C8, 2C9 and 2C19) and CYP4B sub-families of P450's. 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 span a 100-fold range. Those interactions that involve CYP2C9 are pharmacokinetic in origin. Currently, we are adopting a pharmacogenomic approach that focuses attention on new (pharmacodynamic) target genes in the blood clotting pathway, e.g. VKORC1.
CYP4B1 metabolizes a host of pro-toxins, including furans, aromatic amines and certain fatty acids to reactive intermediated 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. The CYP4 enzymes are also unusual because of a novel covalent link that attaches the prosthetic heme group covalently to the protein backbone. We are interested in (i) defining the exact chemical nature of reactive intermediates generated by this enzyme, (ii) characterizing what appears to be an evolutionary defect that renders human CYP4B1 functionally defective and (iii) understanding the structural and mechanistic bases for the unusual substrate specificity of the enzyme and its novel heme covalent link.
In this work we use protein engineering coupled with conventional protein biochemistry methods for the 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 for analyte quantification, including stable isotope labeling studies and evaluation of structural changes in mutant proteins. We are also embarking on our first knockout animal model in order to study mechanisms of CYP4B1 toxicity in the whole animal. Synthetic chemistry comes into play in the preparation of new substrates, inhibitors and metabolites for the CYP2C and CYP4B enzymes. Our long-term goals are to understand how structure and function are related for these important drug-metabolizing enzymes.
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This page last updated: February 25, 2008