抗激酶区突变耐药的ROS1选择性抑制剂的设计、合成及活性研究

ROS1-oncogene fusion proteins are effective molecular targets for anaplastic lymphoma kinase inhibitor in non-small celll lung cancers, but the mutations in kinase domain of ALK-oncogene fusion proteins are responsible for ALK inhibitor resistance in these cancers.At present, only multiple receptor tyrosine kinase inhibitor with ROS1 inhibitory activity are reported overseas, and no ROS1 inhibitors are found in China. Therefore, it is necessary to find new highly selective ROS1 inhibitors for treatment of patients with abnormal ROS1 activity and anti-ALK drug resistance. In our previous study, we designed and synthesized series of inhibitors of c-Met/ALK, and found that the compound SMU-B had strong ROS1 inhibitory activity. In this study, based on the structure of a lead compound SMU-B, we design and synthesize a series of novel type I and type II inhibitors with a new "DFG-shefited" conformation binding, by using of molecular docking, molecular dynamics simulation and 3D-QSAR. We measure ROS1 kinase phosphorylation,cell fusion protein kinase phosphorylation, and the cell growth activity to determine the biological activity of these compounds. The purpose of our study is to find new drugs with high selectivity and activity of anti-ALK drug resistance induced by kinase domain mutation of ROS1.

ROS1酪氨酸激酶与癌基因融合形成的融合蛋白是治疗非小细胞肺癌等肿瘤的有效分子靶点，但激酶结构域突变是引起耐药并导致治疗失败的主要原因。目前已知的ROS1抑制剂是兼有ROS1抑制活性的多靶点酪氨酸激酶抑制剂，国内尚无ROS1抑制剂报道。因此，急需发现新的高选择性ROS1抑制剂用于ROS1异常和突变耐药性肿瘤的治疗。我们前期设计合成了系列c-Met/ALK抑制剂，并发现化合物SMU-B具有很强的ROS1抑制活性。本项目拟以SMU-B为先导化合物，通过分子对接、分子动力学模拟、3D-QSAR等方法，首次设计和合成系列结构新颖的选择性ROS1Ⅰ型抑制剂和以新的“DFG-shefited”构象结合的Ⅱ型抑制剂；并通过测定抑制ROS1生化和细胞激酶的磷酸化，测定融合蛋白激酶区突变耐药细胞的磷酸化以及耐药细胞存活来评价化合物活性；旨在发现高选择性和抗激酶区突变耐药的ROS1抑制剂。

ROS1与癌基因融合伴侣形成的异常融合蛋白表达与肺癌等恶肿瘤的发生密切相关，是当前肿瘤个体化治疗的重要分子靶点之一。克唑替尼是第一个针对该靶点批准上市的药物，用于治疗ALK和ROS1阳性非小细胞阳性患者，但治疗之后即出现耐药。. 本项目以前期研究的SMU-B为先导化合物，开展了作用模式新颖的Ⅱ型（含非经典“DFG-shefited”和经典“DFG-out”构象结合模式）和Ⅰ型抑制剂的ROS1高选择性和克服激酶区突变耐药的抑制剂的设计与合成。采用基于荧光能量共振转移原理测定激酶活性的试剂盒，优化实验参数与条件，开展化合物对ROS1和其它激酶抑制活性分析。选择高表达ROS1的肺癌细胞株测定化合物的抗增殖和激酶磷酸化抑制的活性；构建高表达野生和突变ROS1癌基因融合蛋白工具细胞株测定化合物抗细胞增殖和激酶磷酸化抑制的活性，评价克服突变耐药的活性。建立了Ⅰ型和Ⅱ型不同结构类型化合物的3D-QSAR模型指导化合物分子设计优化。项目研究共设计合成了新的Ⅰ型和Ⅱ型ROS1化合物186个，其中Ⅰ型抑制剂优选化合物4801204对ROS1激酶、突变ROS1G2032R和突变ROS1L2026M的Ba/F3工具细胞株抑制增殖IC50分别为16.7nM、24.8nM（强于克唑替尼40倍以上）和5.31nM； “DFG-shefited”构象结合模式Ⅱ型抑制剂优选化合物2e对转染野生型ROS1、突变ROS1G2032R和突变ROS1L2026M的Ba/F3工具细胞株抑制增殖IC50分别为137.7nM、104.7nM（强于克唑替尼6倍）和233.9nM；经典“DFG-out”构象结合模式Ⅱ型抑制剂优选化合物14c抑制ROS1的IC50为了365 nM，对ALK的选择性为12倍。上述研究结果为ROS1选择性抑制剂作为分子靶点个体化治疗抗肿瘤创新药物的发现提供了新的理论与实验依据及应用推广价值。