基于Aurora A激酶和MYCN(N-MYC)蛋白双靶点发现抗肿瘤小分子抑制剂

MYCN (N-MYC) is a potential target in cancer therapy. It is over-expressed in a number of different types of cancer, most notably neuroblastoma, but also including rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms' tumour, and small cell lung cancer. Despite its prominence in cancer, it is considered undruggable, as its DNA binding domain is composed of two extended alpha helices with no apparent surfaces for small molecule binding. Therefore, novel strategies for identifying MYC inhibitors would be beneficial both clinically and from a research perspective. MYCN has been reported to be stabilized by Aurora kinase A and is an attractive therapeutic target. It was reported that CD532 can dissociate the Aurora A-MYCN complex and drive degradation of MYCN protein through disrupting the native conformation of Aurora A. Inhibition of the MYCN-Aurora kinase A interaction by CD532 specifically led to proteosomal degradation of MYCN by the ubiquitination pathway. Having the dual ability to block both Aurora kinase A activity as well as MYCN stability, CD532 could serve as a promising MYC-directed treatment in cancers where MYCN is amplified or overexpressed. In both a neuroblastoma xenograft model and a Sonic Hedgehog (SHH) subgroup model of medulloblastoma, CD532 was able to diminish MYCN protein levels, reduce tumor volume, and increase survival in vivo..The aim of this study is to optimize conformation-disrupting inhibitors to more strongly distort the Aurora-A structure and thereby more effectively disrupt the Aurora-A/MYCN complex to block non-enzymatic functions of kinase. Based on the binding mode and cavity feature of CD532-Aurora-A co-crystal structure, 6 kinds of structure skeletons were designed. Their substituents R1 and R2 were arranged and combined to generate a virtual compound library containing almost 5000 compounds. Then, 40 high affinity moleculars as target compounds were selected for chemical synthesis by virtual screening and comprehensive evaluating with Accelys Dicovery Studio 4.1 software. Structure-activity relationships of conformation-disrupting inhibitors were summarized based on a series of biological evaluation assays for guiding further optimization.

MYCN（N-MYC）是与肿瘤发生发展、侵袭转移及耐药密切相关的潜在多功能靶点，但其DNA结合域被认为不具备设计直接靶向MYCN蛋白小分子药物的条件。Aurora A能够保护MYCN免于在有丝分裂中被降解，Aurora A构象干扰剂同时具有抑制Aurora A以及降解MYCN蛋白的双功能，可作为一种非常有前途的MYCN导向的癌症治疗新方法。本课题基于干扰Aurora A构象使Aurora A/ MYCN复合物发生离解而导致MYCN被降解的间接靶向MYCN策略，依据报道的Aurora A与其干扰剂CD532的结合模式，以CD532结构为模板，合理构建定向组合库，通过分子对接及药物化学甄别，选择40个高结合能化合物进行合成以及生物活性评价，基于构效分析，指导进一步结构优化，以期获得2-3个活性更高、类药性质更好、并具有自主知识产权的可同时抑制Aurora A和降解MYCN的抗肿瘤小分子化合物。

MYCN（N-MYC）是与肿瘤发生发展、侵袭转移及耐药密切相关的潜在多功能靶点，但其DNA结合域被认为不具备设计直接靶向MYCN蛋白小分子药物的条件。Aurora A能够保护MYCN免于在有丝分裂中被降解，Aurora A构象干扰剂同时具有抑制Aurora A以及降解MYCN蛋白的双功能，可作为一种非常有前途的癌症治疗新方法。本课题基于报道的Aurora A与其干扰剂CD532的结合模式，设计并合成了三种结构类型共计53个化合物，并通过核磁、质谱等分析手段确证了化合物结构。采用双抗体夹心酶联免疫分析法（ELISA）、Kinase-Glo® 激酶发光检测法评价了化合物对Aurora A以及Aurora B的抑制活性，通过四氮唑盐还原法（MTT法）测定化合物对MYCN高表达的肿瘤细胞的抑制活性，获得6个抗肿瘤活性较高的化合物，其中化合物ZLJ 34在体内活性评价中也获得了较好的抗肿瘤效果，在结肠癌HCT116移植瘤模型中，以100mg/kg剂量给药，瘤体抑制率为73.24%，瘤重抑制率达为70.67%；在乳腺癌MX-1移植瘤模型中，以100mg/kg剂量给药，瘤体抑制率为51.33%，瘤重抑制率为45.21%；在紫杉醇耐药的乳腺癌MX-1/Taxol移植瘤模型中，以100mg/kg剂量给药，瘤体抑制率达到75.13%，瘤重抑制率达到73.87%。该化合物的进一步药效学以及成药性评价正在进一步进行中。