克服T790M突变耐药的新型嘧啶[5,6-d]并杂环类高效选择性EGFR抑制剂的设计、合成及抗肿瘤活性研究

Non-small cell lung cancer (NSCLC) is the most common cause of death among all major types of lung cancers, which could be responded remarkably to small molecule epidermal growth factor receptor (EGFR) inhibitors. However,the T790M mutation in EGFR kinase often causes drug resistance to the inhibtiors. Currently, multiple "New-generation" EGFR inhibitors, such as WZ4002, that can overcome EGFR T790M-mediated resistance in preclinical models, but lacking kinase selectivity and giving rise to a variety of side and toxic effects. In our previous study, we found that the pyrimidine core which contained in "Third-generation" EGFR inhibitors, gave the great differences in three-dimensional space and the distance to 790 amino acid residues before and after among mutation via computer-assisted analysis. Additionally, ATP-binding sites in currently available inhibitors account for only a fraction of the ATP-binding pocket in the EGFR T790M protein and are localized in the right side within the cavity of the whole ATP-binding pocket, it is different from other RTKs. In this project, based on this finding and the amino acid sequence right side of ATP-binding pocket, we plan to design and synthesize a novel series of potent Pyrimido[5,6-d]heterocyclic derivatives with deleting Micheal receptor group as selective EGFR T790M inhibitors. Meanwhile, several inhibitors will be chosen to test the inhibition and selectivity between EGFR T790M and other RTKs, investigating the anti-tumor activity in vitro and in vivo, confirmiung the ligand binding mode and the sites of the EGFR T790M protein through the crystallization of protein-ligand complexes. This project will provide new sites of action and selective and efficient inhibitors targeting EGFR T790M in NSCLC.

非小细胞肺癌（NSCLC）是导致死亡数最高的肺癌类型，EGFR抑制剂可有效治疗NSCLC，但T790M突变常导致抑制剂产生耐药性。新一代抑制剂如WZ4002等，虽可有效逆转T790M耐药，但存在激酶选择性差和毒副作用大等缺陷。我们前期发现嘧啶类抑制剂母核与突变前后的790位残基间存在较大的距离空间差异，且EGFR T790M结合口袋右侧存在特异性的氨基酸序列和结构，相异于其他激酶。本项目中，我们拟基于WZ4002在EGFR T790M结合口袋的作用模式，并针对口袋右侧特有的空间和氨基酸序列设计并合成一类去除Michael受体的新型嘧啶[5,6-d]并杂环类高效选择性抑制剂。测试该类抑制剂的激酶抑制和选择性，表征体内外抗肿瘤活性，并通过蛋白-药物共结晶确证其与EGFR T790M的结合模式和位点。本项目将为以EGFR T790M为靶标的抗NSCLC药物研究提供新的作用位点和新型选择型抑制剂。

非小细胞肺癌（NSCLC）是导致死亡数最高的肺癌类型，EGFR抑制剂可有效治疗NSCLC，但T790M突变常导致抑制剂产生耐药性。现有抑制剂如WZ4002等，虽可有效逆转T790M 耐药，但存在激酶选择性差和毒副作用大等缺陷。我们前期发现嘧啶类抑制剂母核与突变前后的790位残基间存在较大的距离空间差异，而且EGFR T790M 结合口袋右侧存在特异性的氨基酸序列和结构，相异于其他激酶。本项目中，我们基于WZ4002在EGFR T790M结合口袋的作用模式，并针对口袋右侧特有的空间和氨基酸序列设计并合成了两个系列共计52个新型嘧啶类EGFR抑制剂。通过体外激酶筛选实验得到19e、19h、563和564共计四个结构新颖的EGFRT790M高效选择性抑制剂。Western Blot、 MTT、Hoechst染色和流式细胞技术等多种实验方法评价了四个活性抑制剂的体外抗肿瘤活性。实验结果证实上述活性化合物不仅有效抑制EGFRT790M高表达的非小细胞肺癌H1975细胞的增殖、阻滞H1975细胞的细胞周期，而且可以有效抑制抑EGFR及其下游增殖相关的ERK、AKT蛋白的磷酸化，促进肿瘤凋亡。裸小鼠荷H975体内肿瘤模型评价了四个活性化合物的体内抗肿瘤活性，这些化合物均能有效抑制肿瘤生长，且不影响小鼠的体重。此外，药代动力学研究结果证实化合物19e和19h生物利用度均大于35%，具有良好的成药性。小分子药物-蛋白共结晶结果证实，活性化合物563的Micheal受体结构片段可与EGFR蛋白的Cys-797形成共价结合，有效延长药物的作用时间和作用效果。本课题的系列研究为EGFR T790M抑制剂的构效关系研究增加了新的内容，为肺癌靶向治疗研究提供了新的先导化合物。