Affinity-based fluorescence polarization assay for screening molecules acting on insect ryanodine receptors

Abstract

Insect ryanodine receptors constitute calcium ion channels, and are the target of novel diamide insecticides containing two types of phthalic and anthranilic diamides. Ryanodine receptors in Periplaneta americana leg muscle were extracted through homogenization and centrifugation. Based on the potential usefulness of a fluorescent probe identified in a previous study, a fluorescence polarization assay was developed with extracted Periplaneta ryanodine receptors, and this technique was used to test the affinity binding of two types of active diamide compounds. Results showed that anthranilic diamides could replace the binding of the fluorescent probe on the ryanodine receptor, but phthalic analogs, including flubendiamide, did not affect the binding of fluorescent probe. Therefore, the developed fluorescence polarization (FP) assay is an easier and more efficient technique to study the affinity binding of small molecules on fluorescent probe binding sites on ryanodine receptors than the [³H] labeled-based assay.