Screening The Kinase Inhibitor Library Against Echinococcus Granulosus Larvae In Vitro And In Vivo

Objective:(1)To screen kinase library against Echinococcus granulosus protoscoleces and cysts in vitro and in vivo:(2)To establish a viable screening platform for identifying the target molecules in the relative pathways associated with kinases;(3)To confirm one ro two small molecules as drug candidates killing E.granulosus cyst.Methods:(1)A Kinase Inhibitor Library from Selleckchem was selected and used for screening killing efficacy against protoscoleces and echinococcal cysts of E.granulosus in vitro at a concentration of 5 ?M as the first screening procedure..(2)Based on the first screening,these small molecules with killing more than 95%of protoscoleces were selected for further killing assay at drug concentration of 1 ?M.(3)Micro-cysts were generated from protoscoleces and those selected drugs were used for killing these micro-cysts.(4)these drugs having killing micro-cysts in vitro were selected for using mouse infection model to evaluate their drug efficacy,including LD50,side-effects affecting organs,decress of echinococcal cysts,wet weight of cysts and microstructure of the cysts by normal H&E staining and transmission electron microscopy(TEM).Blood tests and tissue section of mouse model were also used for evaluating the side effects of the target drugs.Results:The Kinase Inhibitor Library from Selleckchem comprised of 378(including 59 targets)was used from drug screening against protoscoleces(PSC)and cysts of Echinococcus granulosus.(1)Primary screening at 5?M showed 51 compounds(include 29 targets)having killing efficacy against PSC;(2)Further screening with these effect drugs at at 1 ?M identified 7 compounds having druggable efficacy against PSC.LC50 assay revealed that only 2 compounds,namely S2243 and S2895,exhibited their LC50 value below 2.5 ?M.(3)S2895 and S2243 was carried out for in vitro cultivation at 20 mg/mL,which resulted in the 60%of E.granulosus cysts being physically damaged.TEM assay showed all the cysts had incompleted structures including tegument.TEM also showed nucleoluses were disappeared.(4)For in vivo assay,at a dose of 15 mg/kg of body weight was used for 30 days,that resulted in significant reduction of cyst number and weight,and cyst mortality compared with these in abendazole group.All clinical features including blood function features,electrolytes,values associated with liver and renal function were no significant difference between the drug test groups and normal control groups.There were no significant changes in the weight of heart,liver,lungs,kidney,and spleen.In addition,there was no significant change in liver and kidney pathologyIn terms of side-effect.Conclusion:(1)These results provide evidence showing that kinases are potential targets for drug development against E.granulosus protoscoleces ang larval cysts.(2)Some kinase inhibitors have more effective in term of killing protosocleces and larval cysts than others,indicating different kinase pathways have different role for the parasite survival,which may help for drug development.(3)Drug residues play an important role in drug efficacy against E.granulosus cysts.(4)Degrasyn and Tyrphostin 9 are drug candidates for drug development against echinococcosis.