Light Regulation of Insect Behavior by Azobenzene-modified Flonicamid as Novel Molecular Tools

Photopharmacology, an emerging interdisciplinary field, enables precise modulation of target biological activity by employing light-controllable photoswitchable molecules. The research on nicotinamidase (Naam), a recently identified target molecule for pesticides, remains relatively underexplored. Therefore, based on the structure of its inhibitor Flonicamid, we designed and synthesized a series of photopharmacological ligands (FAB_S) by incorporating a light-controllable azobenzene switch, further investigating their effects on insect behavior and activity. Compound FAB03 exhibited an LC₅₀ value of 32.8 μM before light exposure and 154 μM after light exposure against Aedes albopictus, indicating a 4.7-fold difference in activity. Compound FAB05 exhibited an LC₅₀ value of 0.691 μM before light exposure and 2.85 μM after light exposure against nymphs (1 day old) of Aphis craccivora, indicating a 4.1-fold difference in activity. Additionally, this series of photopharmacological ligands has successfully achieved light-mediated modulation of locomotor behavior in both Drosophila melanogaster and A. albopictus larvae. Fluorescence staining experiments with A. albopictus larvae were conducted to explore potential modes of action of the drug. Calcium content analysis showed that Flonicamid at 20 μg/mL significantly caused an inward Ca²⁺ flux in the ovarian of Spodoptera frugiperda (SF9), and the trans-configurations of the photopharmacological ligands FAB03 and FAB05 also led to a certain increase in the concentration of intracellular Ca²⁺. AlphaFold2 was employed to predict the three-dimensional structure of Naam, and potential binding modes were elucidated through molecular docking studies.