Pyrrole-2 carboxamides - A novel class of insect ryanodine receptor activators.

The ryanodine receptor (RyR) is an intracellular calcium channel critical to the regulation of insect muscle contraction and the target site of diamide insecticides such as chlorantraniliprole, cyantraniliprole and flubendiamide. To-date, diamides are the only known class of synthetic molecules with high potency against insect RyRs. Target-based screening of an informer library led to discovery of a novel class of RyR activators, pyrrole-2-carboxamides. Efforts to optimize receptor activity resulted in analogs with potency comparable to that of commercial diamides when tested against RyR of the fruit fly, Drosophila melanogaster. Surprisingly, testing of pyrrole-2-carboxamides in whole-insect screens showed poor insecticidal activity, which is partially attributed to differential selectivity among insect receptors and rapid detoxification. Among various lepidopteran species field resistance to diamide insecticides has been well documented and in many cases has been attributed to a single point mutation, G4946E, of the RyR gene. As with diamide insecticides, the G4946E mutation confers greatly reduced sensitivity to pyrrole-2-carboxamides. This, coupled with findings from radioligand binding studies, indicates a shared binding domain between anthranilic diamides and pyrrole-2-carboxamides.

Discovery and mode of action of afoxolaner, a new isoxazoline parasiticide for dogs.

Afoxolaner is an isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration. In vitro membrane feeding assay data and in vivo pharmacokinetic studies in dogs established an afoxolaner blood concentration of 0.1-0.2 µg/ml to be effective against both fleas (Ctenocephalides felis) and ticks (Dermacentor variabilis). Pharmacokinetic profiles in dogs following a 2.5mg/kg oral dosage demonstrated uniform and predictable afoxolaner plasma concentrations above threshold levels required for efficacy for more than one month. Dose ranging and a 5-month multi-dose experimental study in dogs, established that the 2.5mg/kg oral dosage was highly effective against fleas and ticks, and produced predictable and reproducible pharmacokinetics following repeated dosing. Mode of action studies showed that afoxolaner blocked native and expressed insect GABA-gated chloride channels with nanomolar potency. Afoxolaner has comparable potency between wild type channels and channels possessing the A302S (resistance-to-dieldrin) mutation. Lack of cyclodiene cross-resistance for afoxolaner was confirmed in comparative Drosophila toxicity studies, and it is concluded that afoxolaner blocked GABA-gated chloride channels via a site distinct from the cyclodienes.

Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides.

Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.