基于克服耐药性的ALK和HDAC双靶点抑制剂的设计合成及生物活性研究

ALK inhibitors have demonstrated clear clinical benefit on ALK-positive non-small cell lung cancer, however, acquired resistance invariably develops. AXL kinase overexpression and hyperphosphorylation caused by epigenetic alterations in miRNAs has been implicated as a mechanism of resistance to ALK inhibitors. And combination of HDAC inhibitor could exhibit cooperation and synergy in AXL kinase overexpression inhibition to overcome acquired resistance to ALK inhibitors. Based on this promising result, novel compound PT-3 with ALK/HDAC dual inhibition activities were creatively designed and synthesized through the principle of pharmacophore integration of ceritinib and SAHA or panobinostat in our previous work. On the basis of this lead compound and the structure characteristics of other HDAC inhibitors, this project aims to design and synthesis sixty novel PT-3 derivatives according to the rational drug design and structure optimization strategy. The biological activities of these compounds are evaluated by the experiments of ALK/HDAC inhibition and cell proliferative activities in vitro and in vivo. The molecular mechanism will be preliminarily explored and the structure-activity relationship will be summarized. The complement of the present project will help us to develop novel and potent ALK/HDAC dual inhibitors which are not prone to develop resistance in ALK positive NSCLC therapy and provide a theoretical basis for the further research of ALK/HDAC dual inhibitors.

ALK抑制剂对ALK阳性非小细胞肺癌的疗效显著，但易产生耐药，其中由miRNAs表观遗传改变引起的AXL激酶的过度表达及磷酸化是ALK抑制剂产生耐药的主要机制之一。HDAC抑制剂能通过抑制耐药肺癌细胞中AXL激酶的异常表达及磷酸化而克服ALK抑制剂的耐药性，两者具有协同增效作用。课题组前期以ALK抑制剂色瑞替尼和HDAC抑制剂SAHA、帕比司他为先导化合物，利用药效团融合策略和计算机辅助药物设计，首次发现了具有ALK/HDAC双重抑制活性的化合物PT-3。本项目拟以其为先导化合物，结合其它HDAC抑制剂的结构特点，进行合理药物设计与结构优化，计划合成60个目标化合物，期望通过ALK/HDAC激酶抑制活性和体内外抗肿瘤活性筛选，获得1-2个抗ALK阳性非小细胞肺癌活性强、不易产生耐药、结构新颖的候选化合物，并研究作用机制，总结构效关系，为ALK/HDAC双靶点抑制剂的深入研究提供理论基础。

为了解决二代ALK抑制剂色瑞替尼易产生耐药性的问题，本课题在分析ALK和HDACs抑制剂构效关系的基础上，采用“拼合”策略，将ALK抑制剂色瑞替尼和HDAC抑制剂SAHA结构中相应的药效团进行拼合，借助计算机辅助药物设计方法，共设计合成了I-II个系列73个目标化合物，在此基础上，对合成的化合物进行了初步的药理活性评价和构效关系研究。结果显示，大部分化合物对ALK和HDAC1都具有较高的抑制活性，其中，化合物I-23对ALK(IC50=2.1nM)和HDAC1(IC50=7.9nM)同时具有强效和平衡的抑制活性，与阳性对照克唑替尼和SAHA相当。更重要的是，I-23对导致色瑞替尼耐药主要原因之一的G1202R基因突变，其抑制IC50为0.4nM，约为色瑞替尼的10倍，说明该化合物相比于色瑞替尼可能更易于与ALKG1202突变型耐药体结合，因而更容易在耐药株中发挥活性。同时，化合物I-23对H2228、SK-N-BE(2)和SH-SY5Y等多种ALK高表达的肿瘤细胞也表现出了较强的增殖抑制活性，可引起肿瘤细胞凋亡和周期阻滞，且活性优于阳性对照色瑞替尼和SAHA。然而，药物代谢动力学实验显示I-23的药物代谢性质不佳。因此，在I-23的结构基础上，我们对其进行进一步的结构修饰获得了II系列衍生物II-26。体内实验显示，II-26(25和100mg/kg）在SK-N-BE(2)裸鼠移植瘤模型上具有显著的抑瘤作用，且优于色瑞替尼（50mg/kg）。初步的作用机制表明，化合物I-23和II-26可以同时抑制ALK和HDACs的信号通路。以上研究成果可以为ALK和HDACs双靶点抑制剂的深入研究提供理论依据和实践依据。