The Cytochrome P450 Family 4 Enzymes: Focus on Inhibition by Formamdioxime Analogues and Metabolism of Vitamin K

Abstract

The studies presented in this dissertation explore Cytochrome P450 family 4 (CYP4) enzyme inhibition and metabolism. The structure-activity relationships between CYP4 enzymes and the potent formamidoxime inhibitor, HET0016, was defined, which led to the development of competitive inhibitors that are selective for CYP4F2 and CYP4F3B and do not inhibit CYP4A11. These CYP4 enzymes are principally responsible for biosynthesis of the signaling eicosanoid, 20-HETE. Additionally, HET0016 was established as a mechanism-based inhibitor of CYP4A11, but not of other CYP4 enzymes. Studies to characterize the kinetic properties of this mechanism based inhibitor and identify a reactive species have shown the inactivation is potent and efficient, does not result in metabolite intermediate complex formation or nitric oxide release, and does not destroy the heme cofactor. A carbodiimide or isocyanate reactive intermediate has been proposed that could result in apo-protein adduction. In the second portion of this thesis, it was established that CYP4F2 and CYP4F11 metabolize vitamin K1 and K2 to their respective ω-hydroxy metabolites, and that CYP4F2 sequentially metabolizes vitamin K to the ω-carboxy metabolites. Microsomal alcohol dehydrogenase/ fatty-aldehyde dehydrogenase (ADH/fALDH) or other P450 enzymes may also play a role in formation of ω-carboxy vitamin K after initial ω-hydroxylation by these CYP4 enzymes. For the first time, the amount of CYP4F11 protein in human liver was quantitated, using a new LC-MS/MS method and it was confirmed that the amount of CYP4F2 protein in human liver correlates with the presence of the common <italic>CYP4F2*3</italic> (V433M) allelic variant. Menaquinone-4 (MK4) is one form of Vitamin K2, and this work has shown that metabolism of MK4 is distinctly different from that of Vitamin K1 (phylloquinone). P450 enzymes other than those in the CYP4 family were shown to ω-hydroxylate MK4 in vitro and two new oxidized microsomal metabolites of MK4 were characterized. This work has laid groundwork for future studies that should establish the differences in the metabolic fate of the various forms of vitamin K in vivo and help clarify a genetic basis for the inter-individual variability in response to these supplements when used as pharmacotherapeutic agents.