Alonso-Fernandez, J.-R.; Stamati, E. C. V.; Drakou, C. E.; Mazavli, M.; Karras, V.; Papachristos, D. P.; Michaelakis, A.; Zographos, S. E.; Perez-Sanchez, H.; Tsitsanou, K. E.

Odorant-binding proteins (OBPs) play central roles in insect chemoreception and represent attractive molecular targets for the discovery of novel behavior-modifying compounds. We report two crystal structures of Anopheles gambiae OBP4: one complexed with the fluorescent probe N-phenyl-1-naphthylamine and a second containing two indole molecules simultaneously bound within the ligand-binding cavity. Structural comparison revealed two binding subsites accommodating the aromatic moieties of 1-NPN or two indole molecules, while identifying Thr58 and Thr70 as key residues in ligand recognition. A structure-based virtual screening campaign was performed against a volatility-filtered library from DrugBank, FooDB, BioNet, and natural-volatiles datasets. Twenty-three ligands were selected for fluorescence competitive binding assays, identifying four AgamOBP4 binders with micromolar affinities (Ki = 2.4-21.8 M). Among them were benzoyl peroxide, an active anti-acne ingredient, Avex-73 (blarcamesine), a clinical-stage drug candidate, and 2-phenylethyl benzoate, a food- and fragrance-associated ester. Additional candidates include O,p'-DDE, a metabolite of the insecticide DDT, and fluorene, a polycyclic aromatic hydrocarbon detected in foods as environmental or processing-derived contaminants. Seven selected compounds were subsequently evaluated against Aedes albopictus in in-vivo behavioral assays, with benzoyl peroxide displaying the highest repellency (RI = 81.8% at 0.2 mg cm-2). AI-based protein-ligand modeling provided insights into ligand recognition and suggested relationships among binding mode, binding affinity, and bioactivity. Collectively, these findings establish the structural basis of ligand recognition by AgamOBP4, highlight the potential of drug-repurposing and food-derived chemical space as sources of next-generation mosquito repellents, and open new opportunities for the development of OBP4-based biosensors for food-quality and environmental monitoring applications.