髓母细胞瘤（MB）是儿童中最常见的脑部恶性肿瘤，其Shh亚型以Hedgehog (Hh)信号通路的连续激活为显著特征，目前的治疗药物多为Smo抑制剂，虽疗效显著，但易在Smo 7TM发生突变而耐药。固醇衍生物通过结合Smo CRD抑制Hh通路活性且在Smo突变模型中仍有效果。因此，寻找Smo CRD抑制剂是抵抗Smo突变耐药的一条有效途径。前期从毛叶藜芦中分离得到3个新生物碱且均显著抑制Hh信号通路活性；通过生物活性和分子对接模拟，发现它们很可能是Smo CRD抑制剂且具有抵抗突变耐药的潜能。本项目拟在前期工作基础上，采用荧光素酶报告基因活性系统引导毛叶藜芦系统地分离和纯化，重点是新型具有Hh信号通路抑制活性的生物碱分离；利用竞争性结合实验和基因突变技术阐明生物碱是否作用于Smo CRD和抵抗Smo突变耐药；并进行体外抗MB活性评价和构效关系研究，为获得抵抗耐药的抗MB药物提供候选化合物。

Medulloblastoma (MB) is the most common malignant brain tumor in children and the Sonic hedgehog (Shh) subgroup is one of the four subgroups in MB, which is characterized by constitutive activation of Hedgehog (Hh) signaling pathway. Over the past decade, Smo inhibitors showed good potency in MB, however, currently due to the Smo 7TM mutations, they are limited by drug resistance. Sterol-related derivatives inhibit Hh signaling pathway by binding the cysteine-rich domain (CRD) of Smo and still remain effective on the Smo mutant models. Therefore, finding some novel Smo CRD inhibitors could be a valid approach to overcome drug-resistance. Three novel steroidal alkaloids, obtained from Veratrum grandiflorum, exhibited significantly inhibition activities in Hh signaling pathway. The preliminary results of biological activities and molecular docking simulation suggested that these alkaloids could be Smo CRD inhibitors and have a great potential on drug-resistance. Based on the previous work, the separation and purification of active steroid alkaloids from V. grandiflorum will be guided by dual-luciferase reporter assay system, focusing on the separation of new or active steroid alkaloids; to investigate the binding sites on Smo and the effect on drug-resistance by competitive binding assay and gene mutation; then the anti-medulloblastoma activities in vitro and structure-activity relationships (SARs) will be evaluated. All research work we do aims to put a foundation for the development of novel anti-medulloblastoma drugs against drug-resistance.