title: A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites.
abstract: Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA;
authors.forename: Jamil A
authors.initials: JA
authors.lastname: Haque
authors.forename: Matthew G
authors.initials: MG
authors.lastname: McDonald
authors.forename: John D
authors.initials: JD
authors.lastname: Kulman
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
mesh_terms: D000438:Alcohols / Q000737:chemistry; D000925:Anticoagulants / Q000737:chemistry; D004318:Doxycycline / Q000737:chemistry; D005164:Factor IX / Q000737:chemistry; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008099:Liver / Q000378:metabolism; D019800:Phenol / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014812:Vitamin K / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014859:Warfarin / Q000031:analogs & derivatives / Q000737:chemistry*
chemical_list: D000438:Alcohols; D000925:Anticoagulants; D014812:Vitamin K; D019800:Phenol; D014859:Warfarin; C065719:10-hydroxywarfarin; D005164:Factor IX; D064417:Vitamin K Epoxide Reductases; D004318:Doxycycline