Institute of Experimental Haematology and Transfusion Medicine and.
Institute of Molecular Medicine, University Clinic Bonn, Bonn, Germany.
Blood Adv. 2018 Mar 27;2(6):691-702. doi: 10.1182/bloodadvances.2017006775.
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.
维生素 K 的还原是由两种酶在体外催化的：维生素 K 2,3-环氧化物还原酶复合物亚基 1（VKORC1）及其同工酶 VKORC1 样 1（VKORC1L1）。在体内，VKORC1 将维生素 K 还原以维持维生素 K 依赖性蛋白质（包括凝血因子）的γ-羧化。口服抗凝剂（OACs）通过抑制 VKORC1 在临床上用于治疗和预防血栓形成。然而，OACs 也抑制 VKORC1L1，先前的研究表明它在体外的细胞内氧化还原平衡中发挥作用。在这里，我们首次报告了各种 OACs 和杀鼠剂在基于细胞的测定中对两种 VKOR 酶的特异性抑制的数据。通过 CRISPR/Cas9 技术敲除各自基因的基因工程 HEK 293T 细胞中，独立研究了对内源性 VKORC1 和 VKORC1L1 的影响。一般来说，4-羟基香豆素和 1,3-茚二酮的剂量反应是酶依赖性的，与 VKORC1 相比，VKORC1L1 的敏感性较低。相比之下，杀鼠剂对两种酶的剂量反应几乎相同。为了解释明显的抑制模式，我们进行了计算机模拟，表明 VKORC1 和 VKORC1L1 具有不同的华法林结合位点。我们确定 VKORC1L1 内质网腔环中的位置 38、42 和 68 的精氨酸残基负责电荷稳定的华法林结合，从而形成与 VKORC1 完全相反的结合口袋。总之，我们的研究结果提供了对 VKORC1 尤其是 VKORC1L1 的结构和分子药物结合的深入了解。
