Novel Anopheles glutamate-gated chloride ion channel binding sites facilitate ivermectin mosquito-lethal effect

Ivermectin is lethal to Anopheles mosquitoes making it a possible malaria control intervention. The primary mode of action of ivermectin occurs when it binds to the glutamate-gated chloride channel (GluCl), allowing for continuous flow of chloride leading to flaccid paralysis and death of the mosquito. In Caenorhabditis elegans , ivermectin is thought to open the GluCl channel when the M2-M3 loop forms Van der Waals bonds with the first sugar ring and aglycone structure of ivermectin. Here we investigate in Anopheles dirus and Anopheles minimus the mosquito-lethal effect of ivermectin (both sugar rings), monosaccharide (one sugar ring), and aglycone (no sugar rings) demonstrating full, partial, and no effect, respectively. The Anopheles GluCl protein sequences were determined and used to a create 3-D structural docking models. The docking models identified a novel binding point with a hydrogen bond forming between the second sugar ring hydroxyl group (4”-OH) and THR304 of the Anopheles GluCl M2-M3 loop. This novel hydrogen bond is possible due to a single substitution in the M2-M3 loop from C. elegans ILE273 to Anopheles THR304. The work presented here improves our understanding of Anopheles GluCl-ivermectin interactions as well as how ivermectin resistance could arise in the future.