Study Of Anti-Cancer Effects And Mechanisms Of Novel Hedgehog Inhibitor L-4

Background:The Hedgehog(Hh)signaling pathway plays an important role in mammalian growth and development,and the abnormal expression of Hh pathway has also been shown to be involved in the development of various cancers.In clinical trials,the inhibition of aberrant expression of Hh pathway has proved to be a promising therapeutic intervention against cancers such as basal cell carcinoma(BCC)and medulloblastoma(MB).In the Hh pathway,Smo protein has been widely selected as a target for inhibiting the abnormal expression of the Hh pathway,and various drugs and inhibitors targeting Smo proteins have been developed.Purpose:Currently,two drugs(Vismodegib,Sonidegib)have been approved for the treatment of basal cell carcinoma,and more inhibitors are in clinical research.However,the compounds that have been marketed have produced strong side effects and drug resistance in clinical treatment.Therefore,it is very important to develop new Hh pathway inhibitors that can overcome drug resistance.Here,our team screened a series of compounds synthesized by Fudan University’s Zhao Weili and Dong Xiaochun’s group to identify a new,well-tolerated,orally active small molecule Hh inhibitor by using a variety of in vitro and in vivo model systems.Method and results:In this study,61 synthetic compounds containing a pyridazine or dimethylpyrazine central backbone were screened as novel Hh signaling inhibitors using the DualLuciferase Assay Reporter System.Among these compounds,L-4 had the strongest inhibitory effect with an IC50 value of 2.23 nM.The Brdu method was used to detect cell proliferation.L-4 had a strong growth inhibitory effect on primary medulloblasts with an IC50 of 31.36 nM,while the IC50 values of the positive drugs Vismodegib and LY-2940680 were 37.65 and 35.5 nM,respectively,indicating L-4.It has a similar proliferation inhibition effect as the positive drug.Western Blot showed that L-4 strongly inhibited Hh pathway in vitro.For primary medulloblastoma and Shh-Light II cells,300 nM L-4 significantly inhibited Gli1 protein and Ptch1 protein.The BODIPYcyclopamine competition binding assay demonstrated that L-4 blocked the Hh pathway by antagonizing Smo.When L-4 concentration reached 1 μM,it could compete to antagonize the binding of 50% of the same concentration of fluorescent cyclopamine to Smo,indicating L-4 and The first generation Hh pathway inhibitor cyclopamine has similar ability to bind Smo protein.Significantly,L-4 significantly inhibited the Hh pathway activity induced by the Smo mutant(D473H),which acted on Shh-Light II cells transfected with wild-type and mutant Smo plasmids with IC50 values of 23.46 nM,respectively.It is basically the same as 24.55 nM.The IC50 values of the marketed drug Vismodegib for Shh-Light II cells transfected with wild-type and mutant Smo plasmids were 21.63 and 326.8 nM,respectively,with a difference of 15 times.Molecular docking experiments also validated the results of in vitro anti-drug resistance experiments.In animal experiments,oral administration of L-4 showed significant dose-dependent anti-tumor efficacy in the Ptch+/-;p53-/-transgenic medulloblastoma allograft model,with a minimum dose of 5 mg/kg.There is a significant anti-tumor proliferation effect.After oral administration of 20 mg/kg of L-4,the tumor almost stopped growing,and the therapeutic effect was comparable to that of the positive drug LY-2940680.In addition,L-4 showed good tolerance in the acute toxicity test using ICR mice,and the one-time oral dose of 2000 mg/kg did not produce significant toxic side effects.Conclusion:In this study,the compound L-4 with significant Hh signaling pathway inhibitory activity was screened using the Dual-Luciferase Assay Reporter System.L-4 strongly inhibits the Hh pathway by antagonizing the Smo receptor,and reduces the expression of Gli1 and Ptch1,thereby inhibiting tumor growth.L-4 surpasses current drug use in clinical use.In vivo experiments with the Ptch+/-;p53-/-medulloblastoma transplantation model showed that oral L-4 showed significant anti-tumor efficacy and L-4 showed good tolerance.Our results indicate that L-4 is a novel Hh pathway inhibitor targeting Smo protein,which has the characteristics of inhibiting medulloblastoma activity,tolerance,oral efficacy and resistance.In particular,in terms of overcoming drug resistance,L-4 has a 10-fold higher ability to inhibit mutant Smo protein than the marketed drug at the cellular level,which makes L-4 a Hh inhibitor with better therapeutic effect,which is a promising prospect.Hh targets anti-tumor drugs.If the preclinical and clinical studies of L-4 can be further completed,it is expected to solve the problem of drug resistance and side effects caused by the current marketed drug treatment,and become a new second-line Hh targeted anti-tumor drug that replaces Vismodegib and Sonidiegib.