Virtual screening and activity evaluation of novel ecdysone analogues targeting overlapping binding pockets in Lepidoptera ecdysone receptor.

The ecdysone receptor (EcR/USP) plays a vital role in regulating molting and metamorphosis in insects, rendering it an attractive green target for developing novel insect growth regulators (IGRs). In this study, the ligand pharmacophore model S1&S2 and the Combine active pockets were constructed on the basis of the two different ligand-binding pockets of Lepidoptera EcR. A multi-level virtual screening of the SPECS database was performed, using pesticide-likeness rule and molecular docking, to obtain 13 screening compounds. Then, a novel experimental method was established to evaluate Plutella xylostella EcR/USP-LBD binding to ligand molecules using the Surface Plasmon Resonance (SPR) technique. The binding assays demonstrated that compounds VS-13, VS-16, VS-17, VS-18, and VS-19 exhibited comparable or superior efficacy to the commercial insecticide methoxyfenozide. Notably, VS-13 showed the highest binding activity, with a Kd of 0.2 ± 0.03 μM, similar to Ponesterone A (PonA, Kd = 0.1 ± 0.01 μM). Furthermore, molecular docking studies revealed that, in addition to three important residues (Asn503, Tyr407, and Thr342), Trp525 and Met379 are also key residues in stable the ligand-receptor interactions. These findings provide an effective strategy to design and discovery novel non-ecdysteroid analogs targeting lepidopteran insects.