The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus)

Kazuki Takeda; Yoshinori Ikenaka; Denis Fourches; Kazuyuki D Tanaka; Shouta M M Nakayama; Dhoha Triki; Xinhao Li; Manabu Igarashi; Tsutomu Tanikawa; Mayumi Ishizuka

doi:10.1016/j.pestbp.2021.104774

The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus)

Pestic Biochem Physiol. 2021 Mar:173:104774. doi: 10.1016/j.pestbp.2021.104774. Epub 2021 Jan 9.

Authors

Affiliations

¹ Laboratory of Toxicology, Department of Environmental Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan.
² 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.
³ Department of Chemistry, Bioinformatics Research Center, North Carolina State University, Raleigh, NC, USA.
⁴ Technical Research Laboratory, IKARI SHODOKU CO., Ltd., Narashino, Chiba, Japan.
⁵ Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan; Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Japan.
⁶ Laboratory of Toxicology, Department of Environmental Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan. Electronic address: ishizum@vetmed.hokudai.ac.jp.

PMID: 33771253
DOI: 10.1016/j.pestbp.2021.104774

Abstract

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 IC₅₀ 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_max/K_m). 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.

Keywords: Molecular docking; Molecular dynamics simulations; Rodenticide-resistance; Vitamin K epoxide reductase; Warfarin.

MeSH terms

Animals
Drug Resistance / genetics
Mutation
Rats
Rodenticides* / toxicity
Vitamin K Epoxide Reductases / genetics
Warfarin* / pharmacology

Substances

Rodenticides
Warfarin
VKORC1 protein, rat
Vitamin K Epoxide Reductases