Search

Warfarin is an anticoagulant used for the prevention of thrombosis and embolism. Hydroxylation of warfarin via CYP450s is well documented; however, its biotransformation by UGTs has not been investigated. The activities of 8 human recombinant UGTs, towards warfarin and its hydroxylated metabolites have been assessed with R-warfarin and racemic warfarin, 4′-OH-, 6-OH-, 7-OH-, and 10-OH-warfarin. HPLC-UV-Vis assays and HPLC-MS/MS product confirmations established that UGT1A10, -1A1, -1A3, and -1A8 (highest to lowest activity) conjugated 7-OH-warfarin. Kinetic assays measured Km and Vmax values for 7-OH-warfarin glucuronidation at 165.5 ± 42.7 µM and 1.14 ± 0.1 nmol/mg protein/min, respectively by UGT1A10. 6-OH-warfarin is glucuronidated by 1A1 and 1A10, and 4′-OH-warfarin is glucuronidated by 1A10, but at low levels. Warfarin, 10-OH-warfarin, or R-warfarin were not glucuronidated. This is the first demonstration that major CYP450 metabolites of warfarin are glucuronidated by human recombinant UGTs, with UGT1A10 being the major isoform involved in human phase II biotransformation. [ABSTRACT FROM AUTHOR]

UDP-Glucuronic Acid (UDP-GlcUA) is the common cofactor of all UDP-Glucuronosyltransferase (UGT) isoforms, and it is postulated that its binding site is located within the C-terminal half of the UGT molecule. Amino acid sequence alignment of UDP-glycosyl- and glucuronosyl-transferases as well as other proteins that bind sugars and nucleotides, showed that the DxxD motif in UGTs is a good candidate for the site of UDP binding. Using site-directed mutagenesis and kinetic analysis, we have demonstrated that D393 and D396 of UGT1A10 are involved in the binding of UDP-GlcUA. Mutation of D393 to alanine totally abolished the activity of UGT1A10 toward all substrates analyzed. D396 was also mutated and produced a ∼2 fold decrease in the glucuronidation activity. This mutation did not affect the affinity of this mutant for 2-nitrophenol (pNP); however, its affinity for UDP-GlcUA was decreased by more than half. This is the first direct demonstration that the DxxD motif of UGTs is involved in the binding of UDP-GlcUA. [ABSTRACT FROM AUTHOR]