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Acetylcholinesterase (AChE) regulates nerve signalling and is a well-validated target for insect control in both agriculture and the prevention of mosquito-borne diseases. However, current AChE-targeting insecticides are nonspecific and thus also affect other organisms such as honey bees. The synaptic AChE of honey bees (AChE2) is encoded by the ace-2 gene, thought to have originated from a gene duplication of ace-1. Here, we analyse the structure, dynamics, and kinetics of AChE2 enzymes from the honey bee, Apis mellifera (AmAChE2), and the malaria mosquito, Anopheles gambiae (AgAChE2), and compare them to the more extensively studied type 1 mosquito AChE (AgAChE1) and mammalian AChEs. Important differences between these AChE subtypes were identified. Profiling with selected noncovalent AChE inhibitors revealed strong AChE2 inhibitors, but the inhibition profiles of AChE2 differed substantially from those for AgAChE1 and human AChE. Modelling of AChE2•inhibitor complexes revealed two tyrosines unique to AChE2 that are responsible for these differences in inhibitor sensitivity. These results highlight the importance of considering molecular properties of AChE2 when developing AChE1 inhibitors for pest control. Furthermore, the results also suggest that including AChE2 in computer-aided molecular design efforts during the discovery process could be very valuable for reducing risks of off-target effects.

Vector control of mosquitoes with insecticides is an important tool for preventing the spread of mosquito-borne diseases including malaria, dengue, chikungunya, and Zika. Development of active ingredients for insecticides are urgently needed because existing agents exhibit off-target toxicity and are subject to increasing resistance. We therefore seek to develop noncovalent inhibitors of the validated insecticidal target acetylcholinesterase 1 (AChE1) from mosquitoes. Here we use molecular dynamics simulations to identify structural properties essential for the potency of reversible inhibitors targeting AChE1 from Anopheles gambiae (AgAChE1), the malaria-transmitting mosquito, and for selectivity relative to the vertebrate Mus musculus AChE (mAChE). We show that the collective motions of apo AgAChE1 and mAChE differ, with AgAChE1 exhibiting less dynamic movement. Opening and closing of the gorge, which regulates access to the catalytic triad, is enabled by different mechanisms in the two species, which could be linked to their differing amino acid sequences. Inhibitor binding reduced the overall magnitude of dynamics of AChE. In particular, more potent inhibitors reduced the flexibility of the Ω loop at the entrance of the gorge. The selectivity of inhibitors for AgAChE1 over mAChE derives from the positioning of the α-helix lining the binding gorge. Our findings emphasize the need to consider dynamics when developing inhibitors targeting this enzyme and highlight factors needed to create potent and selective AgAChE1 inhibitors that could serve as active ingredients to combat disease-transmitting mosquitoes.

Resistance development in insects significantly threatens the important benefits obtained by insecticide usage in vector control of disease-transmitting insects. Discovery of new chemical entities with insecticidal activity is highly desired in order to develop new insecticide candidates. Here, we present the design, synthesis, and biological evaluation of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1 (AChE1). AChE1 is a validated insecticide target to control mosquito vectors of, e.g., malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus human AChE selectivity with a high potency also for the resistance-conferring mutation G122S; two properties that have proven challenging to combine in a single compound. Structure activity relationship analyses and molecular dynamics simulations of inhibitor protein complexes have provided insights that elucidate the molecular basis for these properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on disease-transmitting mosquitoes. Our findings support the concept of noncovalent, selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future insecticide development.