基于卤键设计、合成新型EGFRT790M选择性抑制剂及其抗非小细胞肺癌研究

The epidermal growth factor receptor (EGFR) has been established as one of the most important therapeutic targets for non-small-cell lung cancer (NSCLC), and several EGFR inhibitors have been used in the clinical treatment for NSCLC. Unfortunately, EGFR is easily to form drug resistance by gene mutation including T790M. The Third-generation EGFR inhibitors, such as WZ4002, that can overcome EGFR T790M-mediated resistance in preclinical models, but lack kinase selectivity and give rise to a variety of side and toxic effects. Halogen bonding has been recognized as a noncovalent intermolecular and recently used in drug-protein interaction and drug design. Our previous study found that halogen bonding play a vital role in ligand recognition and binding between quinazoline compounds and EGFR binding pocket. This work hypothesize that halogen bonding may be applicable for the design of novel selective EGFR T790M inhibitors. In this project, we plan to apply halogen bonding for the design of novel selective EGFR T790M inhibitors with WZ4002 and our previously found inhibitors as the lead compounds, based on that the great difference in three-dimensional space and the distance to 790 amino acid residues before and after mutation. A variety of experiments are designed to investigate their inhibitory activity and selectivity against T790M mutation after chemical synthesis. Further study contains exploring their binding mode with halogen bonding through the crystallization of protein-ligand complexes and demonstrating their anti-cancer pharmacological activity in vitro and in vivo. This study may provide a new strategy for drug discovery of selective inhibitors targeting EGFR T790M.

EGFR分子靶向治疗是非小细胞肺癌（NSCLC）临床用药手段之一，目前已有多个抑制剂上市，但EGFR T790M突变常导致肺癌耐药高发。第三代抑制剂如WZ4002等，虽可有效逆转T790M耐药，但仍存在选择性不高、存在迈克尔受体和毒副作用大等缺陷。卤键是近年来新发现的非共价结合键型。项目组前期发现卤键在喹唑啉类EGFR抑制剂的受体结合中有着重要作用，可能作为设计新型EGFR T790M选择性抑制剂的有效工具。本项目拟以WZ4002和我们前期发现的抑制剂为先导物，利用790位残基突变前后空间距离的变化和差异，应用卤键设计一类去除迈克尔受体的新型EGFR T790M选择性抑制剂；化学合成后测试它们对突变体的抑制活性和选择性，并通过蛋白-药物共结晶探讨其卤键协助的结合模式，然后表征该类化合物在细胞和动物体内的抗肿瘤活性。本项目将为EGFR T790M选择性抑制剂的药物发现提供新的思路和指导。

EGFR分子靶向治疗是非小细胞肺癌（NSCLC）临床用药手段之一，目前已有多个抑制剂上市，但EGFR T790M突变常导致肺癌耐药高发。第三代抑制剂如WZ4002等，虽可有效逆转T790M耐药，但仍存在选择性不高、存在迈克尔受体和毒副作用大等缺陷。本项目中我们分析了WZ4002与EGFR T790M的结合模式并根据EGFR 激酶口袋的特点，设计、合成、并表征了四类2-氨基嘧类化合物，作为潜在的EGFR T790M选择性抑制剂。项目组通过Lance Ultra Assay 激酶活性筛选方法对各类化合物EGFR T790M的抑制活性和选择性进行了初步考察。再通过MTT法评估化合物对两种不同表型的非小细胞肺癌细胞（H1975 和A431）的抗肿瘤增殖活性以及他们对EGFR及其下游信号通路磷酸化的影响。项目组通过四步结构优化的策略发现了两个潜在EGFR T790M选择性抑制剂D5和D8，它们都具有新颖的化学结构并可与EGFR T790M靶向结合。最后项目组在H1975移植瘤动物模型中进一步考察了化合物D5和D8的体内抗肿瘤效果。结果显示相比较空白组，D5和D8给药组均可显著抑制肿瘤的生长。值得注意的是D5和D8给药组均未显示明显的毒性，这意味着他们都具有开发为EGFR T790M选择性抑制剂类药物的潜力。总的来说，本项目设计了一类2,4-二氨基嘧啶类化合物，并对其进行不断的结构优化，使其成为EGFR T790M选择性抑制剂，相比较野生型突变的EGFR它们具有更高的选择性。以上研究表明这类EGFR T790M选择性抑制剂具有进一步开发为治疗非小细胞肺癌药物的潜力。

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