Mechanism Of Action Of Wilforine On Ryanodine Receptor In Mythimna Separata

There is an urgent demand for novel pesticides with high efficacy,low toxicity,and safety in agricultural production as the acceleration of the process of agricultural modernization in our country.The exceptional properties of natural products offer new ideas and approaches for creating new insecticides and targets.Wilforine（WR）is a small molecular alkaloid compound isolated from the root bark of Tripterygium wilfordii.It has many advantages,such as a wide insecticidal spectrum,environmental safety and non-target biosafety.According to our research group’s previous research,the mechanism of action assumes that WR may act on the ryanodine receptor（Ry R）to cause an imbalance of calcium homeostasis in muscle cells,leading to the flaccid paralysis and death of the oriental armyworm（Mythimna separata）,but direct evidence is lacking.Thus,to investigate the molecular target of WR,verify the binding property and function of WR on the target,and confirm the key binding domain of WR on the target,this paper relies on some methodological techniques,including drug affinity responsive target stability（DARTS）,cell biology,calcium imaging,homology modeling and molecular docking,microscale thermophoresis（MST）,and biolayer interferometry（BLI）.Based on the symptoms and differential proteome analysis in M.separata,we speculate on the relationship between calcium signal inhibition induced by WR and the flaccid paralysis and death of M.separata.This study aims to identify the action target of WR and also expects to find new binding sites and new action mechanisms,which are of great significance for revealing the insecticidal mechanism of WR,accelerating the industrialization process of WR,and targeting new pesticide molecules.The main research results are as follows:1.Wilforine has a high affinity with Ry R.（1）DARTS identified a protective band of WR in the sarcoplasmic reticulum（SR）of M.separata,and its abundance was positively correlated with the concentration of WR.Ry R was found to be the primary protein of the band by HPLC-MS.（2）The immunoblotting abundance of Ms Ry R protein co-incubated with WR reduced in the range of 40℃to 60℃,according to a thermal shift experiment.The fact that Ms Ry R abundance dropped at 50℃with the increase in WR dose suggests that WR binds to Ms Ry R.（3）The Ms Ry R protein was purified from sf9 cells,and the MST test revealed that its equilibrium dissociation constant（K_d）with WR was 91.63 M.Based on the affinity of the two molecules,Ry R is the molecular target of WR.2.Wilforine is an antagonist of Ry R.（1）A calcium ion probe（Fluo-3）-fluorescence spectrophotometer was used to measure the extramembranous Ca²⁺concentration of SR in M.separata;WR did not cause an extramembranous Ca²⁺increase in SR.WR also had no significant effect on endoplasmic reticulum Ca²⁺([Ca²⁺]_ER)in HEK293 cells expressing sf Ry R（Spodoptera frugiperda）and r Ry R（Oryctolagus curiculus）.There was no significant effect on the mortality of M.separata 3rd instar larvae with Ry R knockdown.These findings suggest that WR was unable to activate Ry R.（2）WR decreased the amount of calcium release generated by chlorantraniliprole（CT）,an agonist of Ry R,according to measurements of cytoplasmic Ca²⁺concentration([Ca²⁺]_i)in cultured nerve cells and eukaryotic sf9 cells expressing Ms Ry R,and the weakening effect became more pronounced with the increase in WR’s incubation dose.WR also considerably reduced CT-induced calcium release in SR with a considerable dose-and time-dependent effect.The IC₅₀of WR for suppressing CT-induced calcium release was roughly 339.1μM.Compared with CT alone,CT mixed with WR（50.0μM）showed less stomach toxicity to 3rd instar larvae,and the LC₅₀values rose from 1.27,0.83,and 0.30μg/m L to 6.36,4.45,and 2.82μg/m L after 48,72,and 96 h.These results suggest that WR has an antagonistic impact on Ry R.（3）WR（100.0μM）shifted the dose-effect curve of CT-induced calcium release to the right,and the maximum effect value decreased.As a result,Ry R is the target of WR in terms of the functional influence of drugs on target proteins,and WR interferes with the binding of agonists to Ry R through antagonism.3.The binding domain of wilforine on Ry R is NTD.（1）The three-dimensional structure of M.separata NTD（Ms NTD）,a key domain of Ry R protein,was created using the N-terminal domain（NTD）of rabbit Ry R1 as a homologous model.According to a molecular docking investigation,WR can establish hydrogen bonds with Asn43 in the Ms NTD A subunit,Arg309 in the B subunit,Arg412,Ser416 and Lys420 in the C subunit.（2）As a potential target domain,the equilibrium dissociation constant（K_d）of Ms NTD with WR after prokaryotic expression and purification was 62.84μM（MST）and 61.29μM（BLI）,respectively,indicating that WR and Ms NTD possessed binding properties.These findings indicate that Ms NTD is the essential domain for WR binding to Ms Ry R.4.The mechanism of action of wilforine is related to the downstream effect of calcium the signal caused by inhibiting Ry R.（1）Symptomatological analysis revealed that the poisoning M.separate manifested flaccid paralysis,the transient loss of feeding and crawling ability,and feces retention in the anal opening after treatment with WR（250.0μg/m L）for 48 h.M.separate after receiving CT（100.0μg/m L）treatment for 48 h displayed distinctly contraction,loss of feeding and crawling ability,severe vomiting,and a mortality of 83.96%.WR can alleviate the toxic symptoms of M.separate caused by CT,such as the contraction and vomiting were significantly weakened,and the mortality was reduced by 33.96%.（2）Using the i TRAQ-LC-MS technique,283 differentially expressed proteins in M.separata were identified between the WR and control treatments,including 104 up-regulated and 179down-regulated proteins.Bioinformatics analysis showed that muscle contraction and relaxation-related proteins were involved in the downstream roles of the calcium pathway.In addition,calreticulin,c AMP response element binding protein,and proteins related to ATP synthesis were also down-regulated.The results were consistent with the proteome results obtained by RT-q PCR.It is clear that the divergent toxic symptoms between WR and CT as well as the antagonistic nature of the two insecticidal molecules are connected to their respective effects on Ry R and the subsequent downstream effects of the calcium pathway.In conclusion,WR is an antagonist of Ry R that binds to the Ms NTD domain of Ms Ry R and suppresses Ry R function by allosteric modulation,as demonstrated by our investigations,which established Ry R as the target of WR in terms of binding and function.The proposed mechanism states that Ry R’s conformation is altered by WR binding to NTD,normal intracellular calcium signaling is inhibited,genes or proteins involved in muscle contraction/relaxation and ATP synthesis are downregulated,resulting in damaged muscle cells,as well as the flaccid paralysis and death of M.separate.This study has significant theoretical relevance and practical application guiding value for evaluating the insecticidal molecular mechanism of wilforine,directing the creation of antagonistic compounds targeting Ry R,and encouraging the development and scientific usage of wilforine.