Development of an efficient insecticide substrate and inhibitor screening system of insect P450s using fission yeast

Metabolic resistance is one of the most frequent mechanisms of insecticide resistance, characterized by an increased expression of several important enzymes and transporters, especially cytochrome P450s (CYPs). Due to the large number of P450s in pests, determining the precise relationship between these enzymes and the insecticide substrates is a challenge. Herein, we developed a luminescence-based screening system for efficient identification of insecticide substrates and insect P450 inhibitors. We recombinantly expressed Bemisia tabaci CYP6CM1vQ (Bt CYP6CM1vQ) in the fission yeast Schizosaccharomyces pombe and subsequently permeabilized the yeast cells to convert them into "enzyme bags". We exploited these enzyme bags to screen the activity of twelve luciferin substrates and identified Luciferin-FEE as the optimal competing probe that was further used to characterize the metabolism of eight candidate commercial insecticides. Among them, Bt CYP6CM1vQ exhibited notable activity against pymetrozine and imidacloprid. Their binding modes were predicted by homology modeling and molecular docking, revealing the mechanisms of the metabolism. We also tested the inhibitory effect of eight known P450 inhibitors using our system and identified letrozole and 1-benzylimidazole as showing significant activity against Bt CYP6CM1vQ, with IC₅₀ values of 23.74 μM and 1.30 μM, respectively. Their potential to be developed as an insecticide synergist was further proven by an in vitro toxicity assay using imidacloprid-resistant Bemisia tabaci. Overall, our luciferin-based enzyme bag method is capable of providing a robust and efficient screening of insect P450 substrates and, more importantly, inhibitors to overcome the resistance.