The long-term aim of this research is to understand how human genetic variation influences drug toxicity. In this renewal application, we will continue our efforts to elucidate mechanisms underlying adverse drug reactions to oral anticoagulants, specifically the coumarin drug, warfarin, which is taken daily by over 2 million Americans, and remains one of the most common causes of emergency room visits because of adverse reactions. In the previous funding period the focus was primarily on genetic variation within the cytochrome P450 enzyme, CYP2C9, because it controls metabolic clearance of the more potent (S) enantiomer of the racemic drug. However, during the previous granting period the gene for the warfarin target protein - VKORC1 - was cloned and has become the new focus of this application because it controls the pharmacodynamic response to the drug. In the present application we are attempting to understand;1) regulatory mechanisms that influence vitamin K epoxide reductase (VKOR) hepatic expression and, 2) the fundamental nature of the warfarin binding site(s) in VKOR. To address these goals we have formulated the following aims:
Specific Aim 1 : Determine the functional significance of regulatory (promoter and intronic) polymorphisms at the VKORC1 locus using reporter constructs, site-directed mutagenesis and DNA/protein binding assays.
Specific Aim 2 : Define structure-activity relationships at the level of both the ligand and the protein for reversible and irreversible inhibition of VKOR using a set of structurally diverse vitamin K antagonists together with novel modeling of this membrane-bound enzyme. Due to the broad scope of the proposed research, this proposal brings together investigators at performance sites in Seattle and Milwaukee to work on these problems. Successful completion of these studies will add to our knowledge of the fundamental mechanisms by which;(i) polymorphisms in the VKORC1 gene affect patient dosing with warfarin and (ii) the vitamin K cycle is inhibited by warfarin.
Warfarin is the most widely-used oral anticoagulant in the world, but therapy is difficult to manage because of wide inter-individual variability in response to the drug, a low therapeutic index, and the resulting serious (sometimes fatal) consequences of drug-drug and drug-gene interactions. Genetic variation in VKOR the target enzyme for warfarin's action is the major determinant of warfarin response, but the underlying mechanisms are obscure. Successful completion of the studies proposed herein will improve public health by advancing the era of 'personalized'medicine through an understanding of the fundamental mechanism of action of the widely used anticoagulant and the molecular mechanism(s) by which common polymorphisms in the VKORC1 gene alter liver concentrations of the protein and govern the dose of the drug that is required by patients.
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