Development Of Fluorescence-Based High-Throughput Screening Assay For Identification Of RyR-Targeting Insecticides

Ryanodine receptors（RyR）are large Ca²⁺-release channels located on the endoplasmic reticulum（ER）and sarcoplasmic reticulum（SR）membranes.They are tetrameric molecules composed of four identical subunits.There are four N-terminal regions that extend into the cytoplasm,the C-terminal is a transmembrane domain,and the N-terminal is a hydrophobic domain.RyR is one of the largest ion channel proteins discovered so far,with a molecular weight of around 2.2 MDa.RyR is a ligand-gated channel.In the process of muscle contraction,the action potential will first activate the voltage-gated calcium channel on the cell membrane,causing Ca²⁺influx,opening the RyR,and causing Ca²⁺release from the ER,and it increases the concentration of Ca²⁺in the cytoplasm,thereby causing muscle contraction.Mammal RyR can be divided into three isoforms,RyR1,RyR2,and RyR3.Insects have only one type of RyR.Due to the differences in the structure of RyR in different species,new insecticides that target RyR have a high degree of specificity.Diamide is a class of highly effective and selective insecticides targeting RyR,including Chlorantraniliprole（Chlo）,Flubendiamide and Cyantraniliprole,which can directly bind and activate insect RyR,causing the uncontrolled release of intracellular calcium stores,which eventually leads to exhaustion of calcium stores,muscle spasms and insect death.They are the best-selling insecticide on the market and are registered for controlling Lepidopteran pests.Spodoptera frugiperda（sf）is a pest of the Lepidoptera Noctuidae,which has caused huge agricultural losses in many countries.Due to the massive use of diamide insceticides,the resistance problem of insect has become increasingly prominent,which has caused extremely high drug resistance worldwide and caused a serious resistance crisis.Therefore,it is urgent to develop new green insecticides to deal with the pest crisis.The currently reported insecticide screening is mainly based on the insecticide screening of live insects.The disadvantages of this screening are low throughput,high cost,and poor selectivity.We have developed a fluorescence-based high-throughput assay by using time-lapse fluorescence to measure Ca²⁺concentration change in the ER lumen.We have stably expressed the Ca²⁺fluorescent indicator protein R-CEPIA1er in the ER in HEK293 cells,which can be used to indicate the change of Ca²⁺concentration in the ER and reflect the change of RyR activity.High-throughput screening（HTS）with wildtype Spodoptera frugiperda RyR（WT sf RyR）in 96-plate format was performed with Flexstation 3.0 equipment.The key to HTS with RyR is to be able to quantitatively evaluate the activity of calcium channels under physiological conditions.Through high-throughput screening of the compound library,we identified four new compounds with insecticidal activity,MY-C0001,MY-C0002,MY-C0003,and MY-C0004.Then we compared the species selectivity of these active compounds at the cellular level to ensure that the selectivity for mammals rabbit RyR1（r RyR1）is lower than that for insects.Drosophila melanogaster（fruit fly）,as a model insect,has been widely used in the research of RyR function.We used CRISPR/Cas9 gene editing technology to obtain a Drosophila strain expressing pest RyR,and further confirmed the efficacy of these active compounds on the Drosophila model.The interaction between the active compound and RyR was also analyzed by molecular dynamics simulation（MD）,revealing the binding site and mode of action of the compound from the spatial dimension.Our research has established a fluorescence-based high-throughput screening method for screening active insecticides targeting RyR,bringing new ideas for responding to the insecticide resistance crisis.