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 <b>FAB03</b> exhibited an LC₅₀ value of 32.8 μM before light exposure and 154 μM after light exposure against <i>Aedes albopictus</i>, indicating a 4.7-fold difference in activity. Compound <b>FAB05</b> exhibited an LC₅₀ value of 0.691 μM before light exposure and 2.85 μM after light exposure against nymphs (1 day old) of <i>Aphis craccivora</i>, 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 <i>Drosophila melanogaster</i> and <i>A. albopictus</i> larvae. Fluorescence staining experiments with <i>A. albopictus</i> 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 <i>Spodoptera frugiperda</i> (SF9), and the trans-configurations of the photopharmacological ligands <b>FAB03</b> and <b>FAB05</b> 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.