Study Of Anti-cancer Effects And Mechanisms Of Novel Hedgehog Inhibitor Bxl-7-15

Background:Hedgehog signaling pathway plays a key role in mammalian embryogenesis,and it regulates the growth of the neural tube in the central nervous system.The aberrant activation of Hedgehog signaling pathway leads to abnormal tissue development,and it has been linked to multiple types of malignant tumors,such as the basal cell carcinoma.Research shows that the inhibition of Hedgehog signaling pathway can be a therapeutic method of the related cancers,and up to now,two drugs targeting Hedgehog signaling pathway(Vismodegib and Sonidegib)have been proved for the treatment of basal cell carcinoma.However,severe adverse effects and drug resistance subsequently happened after their clinical application,and novel Hedgehog inhibitors are highly needed to overcome these problems.Our research group cooperated with the group of the School of pharmacy,Fudan University,we synthesized a series of small molecular compounds based on the chemical structure of the current Hedgehog inhibitors,for the development of new antitumor drugs targeting the Hedgehog signaling pathway.Purpose:In this study,we will screen the 61 small molecular compounds developing from the chemical structure of the current Hedgehog inhibitors,and will study the mechanism of the compound Bxl-7-15,which showed the best inhibiting effect of Hedgehog signaling pathway.At last we will evaluate the antitumor effect of the compound Bxl-7-15 in the mouse models of medulloblastoma.Methods:1.We build a dual-luciferase assay model(Shh Light Ⅱ cells)to screen the 61 small molecular compounds,searching for the Hedgehog inhibitors.2.We used the assay of fluorescent BODIPY-cyclopamine competition to detect the binding Site of compound Bxl-7-15.3.We transfected the mutant Smo gene(D473H)into the Shh Light II cells to evaluate the effect of Bxl-7-15 on drug resistance.4.We obtained the Ptch+/-p53+/-mouse by crossing ptcM+/-mice with p53+/-mice,and harvested the primary intracranial medulloblastoma from the Ptch+/-p53+/-mouse.The medulloblastoma was verified by the X-Gal assay.5.We valuated the antitumor effect of the compound Bxl-7-15 in the Ptch+/-p53+/-mouse models of primary intracranial medulloblastoma.Result:1.Among the 61 small molecular compounds,Bxl-7-15 had the best inhibition effect(IC50=2.33 nM).2.Compound Bxl-7-15 binded to the Smo protein.3.Compound Bxl-7-15 could inhibit the Hedgehog signaling pathway of the cells resistant to Vismodegib.4.We obtained the Ptch+/-p53+/-mouse and the primary intracranial medulloblastoma.5.Compound Bxl-7-15 showed in vitro and in vivo anti-tumor efficacy on the medulloblastoma.Conclusion:Our study selected the the Hedgehog inhibitor Bxl-7-15 by using the dual-luciferase assay model.The compound Bxl-7-15 could inhibit the Hedgehog signaling pathway significantly by binding to the Smo protein,and it might be useful for overcoming the drug resistance of Vismodegib.This study had successfully constructed the Ptch+/-p53+/-mouse models of primary intracranial medulloblastomas,and Bxl-7-15 showed significant antitumor effect in the models.Therefore,Bxl-7-15 was a promising antitumor lead compound targeting the Hedgehog signaling pathway,and it was worth further research for overcoming the problem of drug resistance and side effects.Meanwhile,we established the dual-luciferase assay model and the models of medulloblastomas,so that we could screen the inhibitors of Hedgehog,and laid the foundation for the development of the novel Hedgehog inhibitors.