Design,Synthesis And Application Of Fluorescent Probes For Ryanodine Receptor

Ryanodine receptors（Ry Rs）are calcium release channels located on the endoplasmic reticulum membrane,which play important role in excitation-contraction coupling of muscle.Flubendiamide represents a novel chemical family of green insecticides,which selectively activate invertebrate ryanodine receptor（Ry R）by interacting with the receptor distinct from the ryanodine binding site and has almost no effect on mammalian ryanodine receptors.However,their efficacy has been reduced by recently emerged resistant mutations found in field pest populations.Traditional methods to screen Ry R modulators involve either radio-labeled Ry R substrates or calcium signal-based indirect approaches.However,there is lack of Ry R-directed non-isotope molecular tools for Ry R agonists/antagonists screening and biological imaging.We designed a series of fluorescent probes by using the method of computer-aided drug design to elucidate the mechanism of diamide insecticides and to screen new insecticides targeting the ryanodine receptor.Based on the biochemical information of flubendiamide,a representative of diamide insecticides,we constructed a pharmacophore model and designed fluorescent probes.At the same time,the fluorescent probe also needs to conform to the chemical characteristics of flubendiamide,that is,to retain the chemical structure of"diamide".According to the above methods,we designed eight fluorescent probes.Using phthalic anhydride and aliphatic amine as raw materials,7-amino-4-methylcoumarin as fluorescence group,the probe was synthesized by amide coupling method.Among them,probes S5-S8 use chloro-substituted phthalic anhydride as the raw material.Theoretically,there may be two different isomers due to the asymmetric structure of the raw materials.Therefore,we have carried out single crystal diffraction analysis on probes（S5,S6 and S8）to determine the exact structure of the fluorescent probes.In the Ry R localization experiment of the fluorescent probes,we found that the fluorescent probes were enriched on the endoplasmic reticulum of the cells transfected with the insect ryanodine receptor,which realized the co-localization of the fluorescent probes and insect Ry R.In the competitive binding experiment,it was found that compared with the control group,the fluorescence intensity of the cells in the experimental group decreased significantly,and with the increase of the concentration of flubendiamide,the fluorescence intensity of the cells decreased significantly,which proved that the fluorescence probes and flubendiamide had the same binding site of ryanodine receptor.Next,we studied the mechanism of fluorescent probes.In Ry R mediated Ca²⁺transient experiment,we found that six probes can cause the rapid increase of intracellular calcium concentration in the cells transfected with insect ryanodine receptor,while those not transfected did not show the phenomenon of calcium concentration transient.These results show that they can induce the effective release of endoplasmic reticulum calcium in the cells transfected with insect ryanodine receptor,and S1 has a good ability of calcium induction.In addition,in vivo insecticidal activity analysis,three kinds of probes were used to feed the Plutella larvae.The results showed that the probes had certain insecticidal activity against Plutella xylostella larvae,among which S1 resulted in strongest in vivo toxicity to the tested larvae.We believe that these fluorescent probes can be used for high-throughput screening of new insecticides targeting at ryanodine receptor in vivo and in vitro.The appearance of non isotopic ryanodine receptor recognition probes can not only accelerate the screening process of new green pesticides,but also help to decipher the molecular mechanism of high selectivity and resistance of diamide insecticides.