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Johannes Oldenburg - One of the best experts on this subject based on the ideXlab platform.
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VKORC1 and VKORC1l1 have distinctly different oral anticoagulant dose response characteristics and binding sites
Blood Advances, 2018Co-Authors: Katrin J Czogalla, Matthias Watzka, Arijit Biswas, Kerstin Liphardt, Klara Honing, Veit Hornung, Johannes OldenburgAbstract:Vitamin K reduction is catalyzed by 2 enzymes in vitro: the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) and its isozyme VKORC1-like1 (VKORC1L1). In vivo, VKORC1 reduces vitamin K to sustain γ-carboxylation of vitamin K-dependent proteins, including coagulation factors. Inhibition of VKORC1 by oral anticoagulants (OACs) is clinically used in therapy and in prevention of thrombosis. However, OACs also inhibit VKORC1L1, which was previously shown to play a role in intracellular redox homeostasis in vitro. Here, we report data for the first time on specific inhibition of both VKOR enzymes for various OACs and rodenticides examined in a cell-based assay. Effects on endogenous VKORC1 and VKORC1L1 were independently investigated in genetically engineered HEK 293T cells that were knocked out for the respective genes by CRISPR/Cas9 technology. In general, dose-responses for 4-hydroxycoumarins and 1,3-indandiones were enzyme-dependent, with lower susceptibility for VKORC1L1 compared with VKORC1. In contrast, rodenticides exhibited nearly identical dose-responses for both enzymes. To explain the distinct inhibition pattern, we performed in silico modeling suggesting different warfarin binding sites for VKORC1 and VKORC1L1. We identified arginine residues at positions 38, 42, and 68 in the endoplasmatic reticulum luminal loop of VKORC1L1 responsible for charge-stabilized warfarin binding, resulting in a binding pocket that is diametrically opposite to that of VKORC1. In conclusion, our findings provide insight into structural and molecular drug binding on VKORC1, and especially on VKORC1L1.
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two enzymes catalyze vitamin k 2 3 epoxide reductase activity in mouse VKORC1 is highly expressed in exocrine tissues while VKORC1l1 is highly expressed in brain
Thrombosis Research, 2015Co-Authors: Michael Caspers, Matthias Watzka, Katrin J Czogalla, Philipp Westhofen, Kerstin Liphardt, Jens Muller, Johannes OldenburgAbstract:VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways.
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the arg98trp mutation in human VKORC1 causing vkcfd2 disrupts a di arginine based er retention motif
Blood, 2014Co-Authors: Katrin J Czogalla, Matthias Watzka, Simone Rost, Arijit Biswas, Johannes OldenburgAbstract:Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of
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determination of the warfarin inhibition constant ki for vitamin k 2 3 epoxide reductase complex subunit 1 VKORC1 using an in vitro dtt driven assay
Biochimica et Biophysica Acta, 2013Co-Authors: Carville G Bevans, Matthias Watzka, Christoph Krettler, Christoph Reinhart, Helene Tran, Katja Kosmann, Johannes OldenburgAbstract:Abstract Background Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC50), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data. Methods We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (Km) and warfarin IC50 for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC50 to condition-independent Ki. Results Determination of the warfarin Ki specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay. Conclusion The Ki is not equal to the IC50 value directly measured using the DTT-driven VKOR assay. General significance In contrast to warfarin IC50 values determined in previous studies, warfarin inhibition expressed as Ki can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of Ki are required to accurately report and compare in vitro warfarin inhibition results.
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a new cell culture based assay quantifies vitamin k 2 3 epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol driven vkor assay
Journal of Thrombosis and Haemostasis, 2013Co-Authors: Andreas Fregin, C.r. Muller, Matthias Watzka, Simone Rost, Katrin J Czogalla, J Gansler, M Taverna, Carville G Bevans, Johannes OldenburgAbstract:uller CR, Oldenburg J. A new cell culturebased assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay. J Thromb Haemost 2013; 11: 872‐80. Summary. Background: Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT–VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance. Objectives: A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins. Methods: Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin. Results and conclusions: Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nM, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culturebased assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.
Virginie Lattard - One of the best experts on this subject based on the ideXlab platform.
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Structural Insights into Phylloquinone (Vitamin K1), Menaquinone (MK4, MK7), and Menadione (Vitamin K3) Binding to VKORC1
Nutrients, 2019Co-Authors: Nolan Chatron, Etienne Benoit, Abdessalem Hammed, Virginie LattardAbstract:Vitamin K family molecules-phylloquinone (K1), menaquinone (K2), and menadione (K3)-act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1's ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K's hydrophobic tail size and membrane interactions.
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Adaptative evolution of the VKORC1 gene in Mus musculus domesticus is influenced by the selective pressure of anticoagulant rodenticides
Ecology and Evolution, 2017Co-Authors: Joffrey Goulois, Etienne Benoit, Véronique Lambert, Lionel Legros, Virginie LattardAbstract:Anticoagulant rodenticides are commonly used to control rodent pests worldwide. They specifically inhibit the vitamin K epoxide reductase (VKORC1), which is an enzyme encoded by the VKORC1 gene, involved in the recycling of vitamin K. Therefore, they prevent blood clotting. Numerous mutations of VKORC1 gene were reported in rodents, and some are involved in the resistant to rodenticides phenotype. Two hundred and sixty-six mice tails were received from 65 different locations in France. Coding sequences of VKORC1 gene were sequenced in order to detect mutations. Consequences of the observed mutations were evaluated by the use of recombinant VKORC1. More than 70% of mice presented VKORC1 mutations. Among these mice, 80% were homozygous. Contrary to brown rats for which only one predominant VKORC1 genotype was found in France, nine missense single mutations and four double mutations were observed in house mice. The single mutations lead to resistance to first-generation antivitamin K (AVKs) only and are certainly associated with the use of these first-generation molecules by nonprofessionals for the control of mice populations. The double mutations, probably obtained by genetic recombination, lead to in vitro resistance to all AVKs. They must be regarded as an adaptive evolution to the current use of second-generation AVKs. The intensive use of first-generation anticoagulants probably allowed the selection of a high diversity of mutations, which makes possible the genetic recombination and consequently provokes the emergence of the more resistant mutated VKORC1 described to date.
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comparative inhibitory effect of prenylated coumarins ferulenol and ferprenin contained in the poisonous chemotype of ferula communis on mammal liver microsomal VKORC1 activity
Phytochemistry, 2015Co-Authors: Mariesophie Louvet, Gilbert Gault, Manon Boulven, Florence Popowycz, Sébastien Lefebvre, Virginie Lattard, Stéphane Besse, Etienne Benoit, Denis GrancherAbstract:Two distinguishable chemotypes of Ferula communis have been described: the 'nonpoisonous' chemotype, containing as main constituents the daucane esters; and the 'poisonous' chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to 'poisonous' chemotypes of F. communis.
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VKORC1 mutations detected in patients resistant to vitamin K antagonists are not all associated with a resistant VKOR activity
Journal of Thrombosis and Haemostasis, 2012Co-Authors: Ahmed Hodroge, Isabelle Fourel, B. Matagrin, C. Moreau, A. Hammed, E. Benoit, Virginie LattardAbstract:. Background: The VKORC1 gene codes for the VKORC1 enzyme, which is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). Twenty-eight rare single mutations in the VKORC1 coding sequence have been reported from resistant patients receiving unusually high doses of VKA to achieve therapeutic anticoagulation. Objectives: It has been suggested that these mutations are responsible for the resistant phenotype, while biochemical consequences of these mutations on the VKORC1 enzyme have not yet been evaluated. Therefore, the aim of this study was to investigate the causality of the VKORC1 mutations in the resistance phenotype. Methods: Wild-type VKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to VKA was assessed by the in vitro determination of kinetic and inhibition constants. Results and Conclusions: The in vitro analysis revealed that six mutations only (A26P, A41S, V54L, H68Y, I123N and Y139H) were associated with increase in Ki, suggesting their involvement in the resistance phenotype observed in patients. A41S and H68Y led to selective resistance, respectively, to indane-1,3-dione and 4-hydroxycoumarine derivatives. The other mutations did not increase the Ki. Furthermore, 10 mutations (S52L, S52W, W59L, W59R, V66M, V66G, G71A, N77S, N77T and L128R) led to an almost complete loss of activity. These results suggest the existence of other resistance mechanisms.
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Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ahmed Hodroge, Etienne Benoit, Christiane Longin-sauvageon, Isabelle Fourel, Virginie LattardAbstract:Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorcl gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.
Matthias Watzka - One of the best experts on this subject based on the ideXlab platform.
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VKORC1 and VKORC1l1 have distinctly different oral anticoagulant dose response characteristics and binding sites
Blood Advances, 2018Co-Authors: Katrin J Czogalla, Matthias Watzka, Arijit Biswas, Kerstin Liphardt, Klara Honing, Veit Hornung, Johannes OldenburgAbstract:Vitamin K reduction is catalyzed by 2 enzymes in vitro: the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) and its isozyme VKORC1-like1 (VKORC1L1). In vivo, VKORC1 reduces vitamin K to sustain γ-carboxylation of vitamin K-dependent proteins, including coagulation factors. Inhibition of VKORC1 by oral anticoagulants (OACs) is clinically used in therapy and in prevention of thrombosis. However, OACs also inhibit VKORC1L1, which was previously shown to play a role in intracellular redox homeostasis in vitro. Here, we report data for the first time on specific inhibition of both VKOR enzymes for various OACs and rodenticides examined in a cell-based assay. Effects on endogenous VKORC1 and VKORC1L1 were independently investigated in genetically engineered HEK 293T cells that were knocked out for the respective genes by CRISPR/Cas9 technology. In general, dose-responses for 4-hydroxycoumarins and 1,3-indandiones were enzyme-dependent, with lower susceptibility for VKORC1L1 compared with VKORC1. In contrast, rodenticides exhibited nearly identical dose-responses for both enzymes. To explain the distinct inhibition pattern, we performed in silico modeling suggesting different warfarin binding sites for VKORC1 and VKORC1L1. We identified arginine residues at positions 38, 42, and 68 in the endoplasmatic reticulum luminal loop of VKORC1L1 responsible for charge-stabilized warfarin binding, resulting in a binding pocket that is diametrically opposite to that of VKORC1. In conclusion, our findings provide insight into structural and molecular drug binding on VKORC1, and especially on VKORC1L1.
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two enzymes catalyze vitamin k 2 3 epoxide reductase activity in mouse VKORC1 is highly expressed in exocrine tissues while VKORC1l1 is highly expressed in brain
Thrombosis Research, 2015Co-Authors: Michael Caspers, Matthias Watzka, Katrin J Czogalla, Philipp Westhofen, Kerstin Liphardt, Jens Muller, Johannes OldenburgAbstract:VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways.
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the arg98trp mutation in human VKORC1 causing vkcfd2 disrupts a di arginine based er retention motif
Blood, 2014Co-Authors: Katrin J Czogalla, Matthias Watzka, Simone Rost, Arijit Biswas, Johannes OldenburgAbstract:Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of
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human VKORC1 mutations cause variable degrees of 4 hydroxycoumarin resistance and affect putative warfarin binding interfaces
Blood, 2013Co-Authors: Katrin J Czogalla, C.r. Muller, Matthias Watzka, Arijit Biswas, Annchristin Wendeln, Philipp Westhofen, J OldenburgAbstract:Since the discovery of warfarin-sensitive vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), 26 human VKORC1 (hVKORC1) missense mutations have been associated with oral anticoagulant resistance (OACR). Assessment of warfarin resistance using the “classical” dithiothreitol-driven vitamin K 2,3-epoxide reductase (VKOR) assay has not reflected clinical resistance phenotypes for most mutations. Here, we present half maximal inhibitory concentrations (IC 50 ) results for 21 further hVKORC1 mutations obtained using a recently validated cell-based assay ( J Thromb Haemost 11(5):872). In contrast to results from the dithiothreitol-driven VKOR assay, all mutations exhibited basal VKOR activity and warfarin IC 50 values that correspond well to patient OACR phenotypes. Thus, the present assay is useful for functional investigations of VKORC1 and oral anticoagulant inhibition of the vitamin K cycle. Additionally, we modeled hVKORC1 on the previously solved structure of a homologous bacterial enzyme and performed in silico docking of warfarin on this model. We identified one binding site delineated by 3 putative binding interfaces. These interfaces comprise linear sequences of the endoplasmic reticulum–lumenal loop (Ser52-Phe55) and the first (Leu22-Lys30) and fourth (Phe131-Thr137) transmembrane helices. All known OACR-associated hVKORC1 mutations are located in or around these putative interfaces, supporting our model.
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determination of the warfarin inhibition constant ki for vitamin k 2 3 epoxide reductase complex subunit 1 VKORC1 using an in vitro dtt driven assay
Biochimica et Biophysica Acta, 2013Co-Authors: Carville G Bevans, Matthias Watzka, Christoph Krettler, Christoph Reinhart, Helene Tran, Katja Kosmann, Johannes OldenburgAbstract:Abstract Background Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC50), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data. Methods We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (Km) and warfarin IC50 for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC50 to condition-independent Ki. Results Determination of the warfarin Ki specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay. Conclusion The Ki is not equal to the IC50 value directly measured using the DTT-driven VKOR assay. General significance In contrast to warfarin IC50 values determined in previous studies, warfarin inhibition expressed as Ki can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of Ki are required to accurately report and compare in vitro warfarin inhibition results.
Etienne Benoit - One of the best experts on this subject based on the ideXlab platform.
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Structural Insights into Phylloquinone (Vitamin K1), Menaquinone (MK4, MK7), and Menadione (Vitamin K3) Binding to VKORC1
Nutrients, 2019Co-Authors: Nolan Chatron, Etienne Benoit, Abdessalem Hammed, Virginie LattardAbstract:Vitamin K family molecules-phylloquinone (K1), menaquinone (K2), and menadione (K3)-act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1's ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K's hydrophobic tail size and membrane interactions.
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Adaptative evolution of the VKORC1 gene in Mus musculus domesticus is influenced by the selective pressure of anticoagulant rodenticides
Ecology and Evolution, 2017Co-Authors: Joffrey Goulois, Etienne Benoit, Véronique Lambert, Lionel Legros, Virginie LattardAbstract:Anticoagulant rodenticides are commonly used to control rodent pests worldwide. They specifically inhibit the vitamin K epoxide reductase (VKORC1), which is an enzyme encoded by the VKORC1 gene, involved in the recycling of vitamin K. Therefore, they prevent blood clotting. Numerous mutations of VKORC1 gene were reported in rodents, and some are involved in the resistant to rodenticides phenotype. Two hundred and sixty-six mice tails were received from 65 different locations in France. Coding sequences of VKORC1 gene were sequenced in order to detect mutations. Consequences of the observed mutations were evaluated by the use of recombinant VKORC1. More than 70% of mice presented VKORC1 mutations. Among these mice, 80% were homozygous. Contrary to brown rats for which only one predominant VKORC1 genotype was found in France, nine missense single mutations and four double mutations were observed in house mice. The single mutations lead to resistance to first-generation antivitamin K (AVKs) only and are certainly associated with the use of these first-generation molecules by nonprofessionals for the control of mice populations. The double mutations, probably obtained by genetic recombination, lead to in vitro resistance to all AVKs. They must be regarded as an adaptive evolution to the current use of second-generation AVKs. The intensive use of first-generation anticoagulants probably allowed the selection of a high diversity of mutations, which makes possible the genetic recombination and consequently provokes the emergence of the more resistant mutated VKORC1 described to date.
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comparative inhibitory effect of prenylated coumarins ferulenol and ferprenin contained in the poisonous chemotype of ferula communis on mammal liver microsomal VKORC1 activity
Phytochemistry, 2015Co-Authors: Mariesophie Louvet, Gilbert Gault, Manon Boulven, Florence Popowycz, Sébastien Lefebvre, Virginie Lattard, Stéphane Besse, Etienne Benoit, Denis GrancherAbstract:Two distinguishable chemotypes of Ferula communis have been described: the 'nonpoisonous' chemotype, containing as main constituents the daucane esters; and the 'poisonous' chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to 'poisonous' chemotypes of F. communis.
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Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants
Archives of Biochemistry and Biophysics, 2011Co-Authors: Ahmed Hodroge, Etienne Benoit, Christiane Longin-sauvageon, Isabelle Fourel, Virginie LattardAbstract:Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorcl gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.
Simone Rost - One of the best experts on this subject based on the ideXlab platform.
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the arg98trp mutation in human VKORC1 causing vkcfd2 disrupts a di arginine based er retention motif
Blood, 2014Co-Authors: Katrin J Czogalla, Matthias Watzka, Simone Rost, Arijit Biswas, Johannes OldenburgAbstract:Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of
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confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor ix and in silico protein modelling
BMC Genetics, 2014Co-Authors: Elisabeth Muller, C.r. Muller, Alexander Keller, Andreas Fregin, Simone RostAbstract:VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
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a new cell culture based assay quantifies vitamin k 2 3 epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol driven vkor assay
Journal of Thrombosis and Haemostasis, 2013Co-Authors: Andreas Fregin, C.r. Muller, Matthias Watzka, Simone Rost, Katrin J Czogalla, J Gansler, M Taverna, Carville G Bevans, Johannes OldenburgAbstract:uller CR, Oldenburg J. A new cell culturebased assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay. J Thromb Haemost 2013; 11: 872‐80. Summary. Background: Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT–VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance. Objectives: A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins. Methods: Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin. Results and conclusions: Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nM, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culturebased assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.
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thirteen novel VKORC1 mutations associated with oral anticoagulant resistance insights into improved patient diagnosis and treatment
Journal of Thrombosis and Haemostasis, 2011Co-Authors: Matthias Watzka, C.r. Muller, Simone Rost, Christof Geisen, Erhard Seifried, Carville G Bevans, K Sittinger, Gabriele Spohn, Johannes OldenburgAbstract:Summary. Background: Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. Objectives: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. Patients/Methods: Six hundred and twenty-six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. Results: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26Thr, His28Gln, Asp36Gly, Ser52Trp, Ser56Phe, Trp59Leu, Trp59Cys, Val66Gly, Gly71Ala, Asn77Ser, Asn77Tyr, Ile123Asn, and Tyr139His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29Leu, Asp36Tyr, and Val66Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non-VKORC1*2 and wild-type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane–lumen interface. Conclusions: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.
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novel mutations in the VKORC1 gene of wild rats and mice a response to 50 years of selection pressure by warfarin
BMC Genetics, 2009Co-Authors: Simone Rost, Johannes Oldenburg, Hansjoachim Pelz, Sandra Menzel, Alan D Macnicoll, Vanina Leon, Ki Joon Song, Thomas Jakel, C.r. MullerAbstract:Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control. In the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins Our results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.
