Chalkiadaki, M.; Grigoraki, L.; Tsakireli, D.; Vasalaki, G.; Tzimas, P. S.; Chen, M.; Remadi, L.; Ragno, R.; Akrani, I.; Mikros, E.; Panteleri, R.; Vlogiannitis, S.; Myrianthopoulos, V.; Kostakis, I. K.; Skaltsounis, L. A.; Vontas, J.; Halabalaki, M.

Insecticide resistance in mosquitoes, largely mediated by cytochrome P450 monooxygenases (CYPs), compromises the efficacy of vector control tools. In this study, chemically-wise selected natural extracts and compounds were screened for their CYP inhibition potential. Among 37 tested plant extracts and fractions, a decarboxylated acidic fraction of industrial hemp (Cannabis sativa Linnaeus var. 'Futura 75') emerged as a promising hit, and phytochemical profiling identified cannabidiol (CBD) as its major component (IC50= 18.37 M for CYP9K1). CBD was used as a scaffold to generate semisynthetic analogues; of which a piperazinyl analogue outperformed the natural scaffold demonstrating significantly greater potency (IC50 = 2.50 M for CYP9K1). Docking studies using homology-derived CYP9K1 models also supported a stronger binding affinity of the piperazinyl analogue relative to CBD. Toxicity assays using pyrethroid-resistant Anopheles gambiae Giles adults confirmed that neither CBD nor the piperazinyl analogue had intrinsic toxicity, yet the semisynthetic analogue significantly enhanced deltamethrin efficacy, showing a threefold synergistic effect. The safety profile of the cannabis compounds for non-target organisms was evaluated through human cell line cytotoxicity tests and bee toxicity assays, suggesting low non-target organism toxicity. Our study describes the identification of a plant-derived synergist lead with strong potential as an insecticide additive to combat metabolic resistance in malaria-transmitting mosquitoes.