A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Abstract

• Factor IX glutamyl carboxylation in engineered HEK 293 cells recapitulates in vivo anticoagulant inhibition of vitamin K cycle activity.• Warfarin metabolite structureactivity analysis on vitamin K cycle antagonism determines their contributions to in vivo anticoagulation.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 g-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 g-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular g-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 g-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. (Blood. 2014;123(4):582-589)

VKORC1 and VKORC1L1 have distinctly different oral anticoagulant dose-response characteristics and binding sites

“…14,17,32,33 Alternatively, in the cell-based assays, a reporter protein that requires g-carboxylation for biological activity is used as marker for VKR and VKOR activity. [34][35][36][37] In several studies, WT FIX and chimeric FIX Gla domain-containing proteins were used as reporter for VKOR/VKR activity and were shown to reflect the in vivo situation properly. 34,[36][37][38] Until now, the majority of the studies could not discriminate between VKOR/ VKR activities from endogenously expressed VKOR enzymes.…”

“…[34][35][36][37] In several studies, WT FIX and chimeric FIX Gla domain-containing proteins were used as reporter for VKOR/VKR activity and were shown to reflect the in vivo situation properly. 34,[36][37][38] Until now, the majority of the studies could not discriminate between VKOR/ VKR activities from endogenously expressed VKOR enzymes. Our assay analyzes endogenous inhibition of VKORC1 and VKORC1L1 independently by using genetically engineered VKORC1L1 and VKORC1 KO HEK 293T cell lines, respectively.…”

“…14,17,33,36 Various warfarin IC 50 values, primarily for VKORC1, have been published and range between 25 nM and 24 mM. 13,32,33,[35][36][37] Differences may arise from the type of assay used (DTT vs cell-based), the type of substrate (K 1 vs K 1 .O) and its concentration, and the relative protein expression levels (endogenously vs overexpressed), and source of cells (kidney-vs liver-derived) ( Table 2). We observed that the type of substrate (K 1 or K 1 .O) did not affect warfarin dose responses when using the same molarity (Tables 1; supplemental Table 1).…”

“…The same inhibitory trend of coumarin derivatives regarding VKORC1 was shown by several in vitro and in vivo studies (Table 2). 14,17,[32][33][34][35][36][37] However, most studies relied on overexpressed VKOR enzymes. Tie et al were the 35 However, they detected no difference in inhibition between WT and VKORC1 KO HEK 293 cells, and VKORC1L1 KO cells were not examined.…”

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

“…Recently, similar cell-based assays have been performed using different reporter proteins (Fregin et al, 2013; Haque, McDonald, Kulman, & Rettie, 2014). Haque et al engineered an HEK293-derived reporter cell line (HEK293-C3) that expresses a chimeric reporter protein, F9CH, which is a VKD integral membrane protein (human proline-rich Gla protein 2) with its Gla domain replaced by FIXgla (Haque et al, 2014).…”