Structural and Biological Evidence for Acetylcholinesterase-Targeted Insecticidal Activity in Musca domestica: A 4.5 ...

House fly (Musca domestica) control faces mounting resistance to conventional insecticides, driving interest in novel chemistries targeting acetylcholinesterase (AChE). We examined eight benzenesulfonamide derivatives originally developed as carbonic anhydrase inhibitors to explore their potential as AChE-targeting insecticides and to identify structural determinants of biological activity. Using a high-quality homology model of M. domestica AChE, we performed blind docking, refined docking to the peripheral anionic site, 100 ns molecular dynamics simulations, and molecular mechanics-poisson-boltzmann surface area (MM-PBSA) binding energy calculations. All eight compounds showed favorable binding energies (-8.5 to -10.2 kcal/mol) and stable trajectories, yet bioassays revealed a stark divide: five compounds (16b, 16c, 16d, 19b, and 19f) produced no mortality at 5% concentration, while 14g and 17c achieved complete adult kill at 5% with LC₅₀ values of 1.37% (95% CI: 1.15%-1.63%) and 1.64% (95% CI: 1.38%-1.95%), respectively. The critical difference was geometric, not thermodynamic. Active compounds 14g and 17c penetrated the ~20 Å aromatic gorge to position within 2.5-2.8 Å of the catalytic serine (Ser318), forming hydrogen bonds and halogen bonds with the triad. Inactive compounds arrested at 7.0-7.5 Å, trapped in the peripheral anionic site with negligible catalytic residue contact despite favorable overall binding. This 4.5 Å threshold correlated with biological activity and provided a quantitative filter for prioritizing AChE inhibitor candidates. Compound 14g, bearing 3,4-dichloro substituents, showed the closest approach (2.5 Å) and strongest binding energy (-37.4 kcal/mol), outperforming chlorpyrifos (-32.7 kcal/mol). Importantly, whole-organism mortality does not prove direct AChE inhibition; alternative mechanisms including cuticular penetration, metabolic disruption, or off-target effects cannot be excluded without direct enzymatic assays. These findings suggest that geometric distance to the catalytic triad, not binding energy alone, correlates with biological activity and provides a quantitative filter for prioritizing AChE inhibitor candidates.