可克服T790M和C797S耐药的EGFR变构抑制剂的设计、合成及生物活性评价

Epidermal growth factor receptor (EGFR) is an important target for the treatment of non-small cell lung cancer (NSCLC). The EGFR inhibitors Gefitinib, Erlotinib and Afatinib are approved treatments for NSCLCs harboring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary mutation in exon 20 of the kinase domain consisting in a single amino acid substitution from threonine to methionine at position 790 (T790M) within the ATP site of the receptor. Recently developed mutant selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of Cys797, the cysteine residue with which they form a key covalent bond. While all current EGFR inhibitors target the ATP-site of the kinase, the remarkable sequential and structural similarity among ATP pockets renders the selective inhibition of kinases a daunting challenge, highlighting the need for therapeutic agents with alternative mechanisms of action. . Allosteric inhibitors, targeting the pocket of kinases outside the highly conversed ATP pocket, have been proposed as a promising alternative to overcome current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance.. In our previous study, based on the co-crystal structure of allosteric inhibitor EAI001 and EGFR (T790M), approximately 3,200,000 druggable molecules were screened through the docking simulations and a number of potential inhibitors with high inhibitory activity against the T790M mutant and the selectivity over the wild type were identified, providing a good starting point for the further development of EGFR allosteric inhibitors. . Based on our previous work, we will optimize the represent hit compounds based on the binding mode of the hit compound in complex with T790M mutant. Because the C797S mutation does not significantly alter the structure or ATP binding affinity of the EGFR kinase, these new compounds are expected to overcoming T790M and C797S mutation simultaneously.

表皮生长因子受体（EGFR）是非小细胞肺癌治疗的重要靶点。目前的EGFR抑制剂均为ATP竞争性抑制剂，长期应用会引起获得性耐药，其主要耐药机制为ATP结合位点的T790M突变。新开发第三代抑制剂可以高选择性的抑制T790M突变体，但是C797S突变很快出现，并且是第三代抑制剂耐药的主要机制。与ATP竞争性抑制剂相比，变构抑制剂利用远离ATP结合位点的变构口袋，具有选择性好、活性高等优点，有望克服T790M和C797S突变耐药。前期,本课题组基于变构抑制剂与EGFR（T790M）的复合物晶体结构，进行了高通量虚拟筛选，获得了具有潜力的苗头化合物。本项目将在此基础上，基于苗头化合物的结构骨架以及与EGFR的结合模式，优化苗头化合物，旨在发现能够克服T790M和C797S耐药的高活性、高选择性的变构抑制剂，为获得具有临床应用价值的EGFR抑制剂提供理论与实验依据。

表皮生长因子受体（EGFR）是非小细胞肺癌治疗的重要靶点。目前的EGFR抑制剂均为ATP竞争性抑制剂，长期应用会引起获得性耐药。与ATP竞争性抑制剂相比，变构抑制剂利用远离ATP结合位点的变构口袋，具有选择性好、活性高等优点，有望克服ATP口袋的突变耐药。.在本项目中，通过分子对接、分子动力学和自由能分解等计算手段对EGFR变构抑制剂与激酶的结合机制和模式进行分子模拟，揭示了影响抑制剂活性和选择性的关键氨基酸残基，为化合物的结构优化提供理论依据。基于变构抑制剂与EGFR的结合模式和分子模拟的提示，对虚拟筛选获得的苗头化合物进行结构优化，设计、合成了2个系列结构新颖的化合物，并进行了活性评价。激酶活性评价表明，大部分化合物对激酶EGFR（L858R/T790M）有较好的抑制活性。体外肿瘤细胞增殖抑制活性评价表明，大部分化合物对高表达EGFR（L858R/T790M）的人非小细胞肺癌H1975细胞有较好的增殖抑制活性，高于阳性对照药EAI045，与JBJ-04-125-02相当。部分化合物对Ba/F3-L858R/T790M/C797S细胞有较好的抑制活性。化合物1e，2e，3b，4a，6a，7a，9a，10a，B1-a3和B1-a5对H1975和Ba/F3-L858R/T790M/C797S细胞的抗增殖活性达到μM级别。其中，化合物2b对H1975细胞有最强的抗增殖活性，IC50为0.4μM，对L858R/T790M和L858R/T790M/C797S突变体有较强的抑制活性，IC50分别为0.702nM和6.517nM，但对Ba/F3-L858R/T790M/C797S没有表现出明显的抑制活性；化合物4a对L858R/T790M和L858R/T790M/C797S突变体有较强的抑制活性，IC50分别为0.669nM和3.002nM，对H1975和Ba/F3-L858R/T790M/C797S的细胞有较强的抑制活性，IC50分别为1.16μM和3.12μM，而对A549没有明显的抑制效果，表现出较好的选择性。深入的研究表明，化合物2b的激酶抑制剂活性不会因ATP浓度的变化而改变，为非ATP竞争性激酶抑制剂。化合物2b可以阻滞H1975细胞在G0/G1期，可显著诱导H1975凋亡，且呈剂量依赖性。本项目为开发具有临床应用价值的EGFR变构抑制剂研究提供理论和实验依据。