Takeda Kazuki, Ikenaka Yoshinori, Fourches Denis, Tanaka Kazuyuki D, Nakayama Shouta M M, Triki Dhoha, Li Xinhao, Igarashi Manabu, Tanikawa Tsutomu, Ishizuka Mayumi
Laboratory of Toxicology, Department of Environmental Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan.
Laboratory of Toxicology, Department of Environmental Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan; Water Research Group, Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa.
Pestic Biochem Physiol. 2021 Mar;173:104774. doi: 10.1016/j.pestbp.2021.104774. Epub 2021 Jan 9.
Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138). However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. We conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant. On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (V/K). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Lys30, Phe55, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.
众所周知的4-羟基香豆素衍生物，如华法林，可作为维生素K环氧还原酶（VKOR）的抑制剂，并用作抗凝剂。VKOR酶的突变可导致对这些化合物产生抗性。这一直是将它们用作药物或灭鼠剂时存在的问题。这些突变大多位于内质网腔环结构（Lys30、Phe55）和跨膜螺旋（Tyr138）内潜在的华法林结合位点附近。然而，在东京发现的对华法林耐药的大鼠中的一种VKOR突变并不遵循这种模式（Leu76Pro），其对VKOR功能和结构的影响仍不清楚。我们进行了体外动力学分析和计算机对接研究，以表征VKOR突变体。一方面，与敏感大鼠（S-大鼠）相比，耐药大鼠（R-大鼠）对华法林的IC值增加了37.5倍；另一方面，R-大鼠的基础VKOR活性（V/K）降低了16.5倍。对接计算表明，R-大鼠的突变型VKOR对华法林的亲和力降低。分子动力学模拟进一步揭示，R-大鼠中与VKOR相关的华法林更易暴露于溶剂中，Lys30、Phe55与华法林之间的关键相互作用更不受青睐。本研究得出结论，VKOR第76位的单一突变通过改变两者之间分子间相互作用的类型和数量，导致对华法林产生显著抗性。
