Investigation of mechanisms of rodenticide-resistance using a closed colony of rodenticide-resistant black rats (Rattus rattus) from Tokyo

Chapter I - Introduction - Anti-blood coagulation rodenticides, such as warfarin, have been used since the 1940s. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats all over the world. Thus, it is necessary for development of novel rodenticide to reveal mechanism of the resistance. I investigated the mechanism with a closed colony of warfarin-resistant black rats (Rattus rattus) from Tokyo. Chapter II - Target enzyme: VKOR - Mutations of the VKOR enzyme can lead to resistance to those compounds. 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) of VKOR. 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. Thus, I 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 (Vmax/Km). 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 Phe55, Lys30, 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. Chapter III - Assessment of ADME - Absorption, distribution, metabolism, and excretion (ADME) is kno