高选择性的新型二苯并[b,d]吡喃-6-酮类PI3Kα抑制剂的分子构建及克服NSCLC对EGFR-TKIs获得性耐药的研究

Acquired resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) remains a difficult and major problem. Studies have identified three different acquired resistance mechanisms to EGFR tyrosine kinase inhibitor, a secondary mutation EGFR T790M, amplification of the MET oncogene and expression of IGFR-1. But the current treatment strategies can not take into account three resistance mechanisms. Although excessive activation of PI3Kαis an essential and common pathway of three resistance mechanisms above, current PI3Kαinhibitors exhibit low selectivity among PI3K isoforms (PI3Kα、β、δ、γ) and so have side effects. A new PI3Kαinhibitor which has a novel dibenzo[b,d]pyran-6-one scaffold was discovered previously by us. So based on the previous study, fragment-based drug design approach in which dibenzo [b,d]pyran-6-one scaffold is used as a core will be used to design new compounds according to different amino acid of PI3K isoforms in catalytic region. Through chemical synthesis and kinase assay in vitro, novel PI3Kαinhibitors which are highly selective will be discovered. Three-dimensional quantitative structure-activity relationship approach will be used to discover the new structural characteristics of selective PI3Kαinhibitors. Finally the models in vitro and vivo and Western Blot will be used to identify that selective PI3Kαinhibitor can overcome all three acquired resistance mechanisms to EGFR tyrosine kinase inhibitor by inhibiting activation of PI3Kαin NSCLC. Therefore this study will lay the foundation for the discovery of highly efficient, lowly toxic candidate drug which is used to NSCLC chemotherapy, and provide a new molecular targeted therapy for the treatment of acquired resistance to EGFR-TKIs in NSCLC.

EGFR-TKIs获得性耐药是NSCLC治疗中的难题。EGFR二次突变、MET扩增和IGFR-1表达是其主要机制，但目前的治疗策略无法兼顾三种耐药机制。PI3Kα过度活化是三种耐药机制共有的、必需的关键途径，但现有的PI3Kα抑制剂选择性低，副作用大。项目前期发现全新结构类型-二苯并[b,d]吡喃-6-酮类的PI3Kα抑制剂。本项目拟以二苯并[b,d]吡喃-6-酮为母核，以PI3K各亚型催化区域差异氨基酸为作用受点，采用基于碎片结构的药物设计技术构建新化合物，经化学合成和激酶活性测定，发现高选择性、全新结构的PI3Kα抑制剂，并揭示其新的结构特性；采用体外、体内及Western Blot方法，揭示PI3Kα选择性抑制剂兼顾克服三种耐药机制的作用。本项目旨在为发现全新结构的高效、低毒的NSCLC化学治疗侯选药物奠定基础；为EGFR-TKIs获得性耐药NSCLC的治疗提供新的分子靶向治疗方法。

本项目以二苯并[b,d]吡喃-6-酮为母核结构，采用基于靶点的计算机辅助药物设计方法，设计并合成了104个二苯并[b,d]吡喃-6-酮类化合物，其结构均经1H-NMR和MS确证。经PI3K激酶活性测定，发现了新型二苯并[b,d]吡喃-6-酮类PI3Kα抑制剂，揭示了该类抑制剂的构效关系及结构特性。. 采用分子对接和分子动力学模拟方法，研究PI3Kα与其选择性抑制剂A66S、Liphagal的作用模式，发现ATP结合区域的C末端部分的Gln859是A66S结构中L-脯氨酰胺部分的选择性作用位点，p-loop环的Met772可能是Liphagal具有选择性的关键因素。在此基础上，设计合成了29个含有L-脯氨酰胺部分的化合物，发现了新结构的PI3Kα抑制剂化合物127，其对PI3Kα的选择性和对PI3Kα过度活化的肿瘤细胞的增殖抑制活性均与国外报道的PI3Kα抑制剂BYL-719相当。. 吉非替尼与化合物127浓度比为1:10时对EGFR T790M二次突变NSCLC细胞H1975的增殖抑制具有明显的协同作用，可以提高耐药的NSCLC细胞凋亡，抑制NSCLC细胞克隆形成和迁移，化合物127可以克服EGFR T790M二次突变导致的吉非替尼获得性耐药。化合物127抑制Akt308和Akt473的磷酸化，阻断PI3K/Akt通路是其克服EGFR T790M二次突变导致的吉非替尼获得性耐药的机制。. 在项目执行过程中，课题组意外发现二苯并[b,d]吡喃-6-酮类化合物51对结肠癌细胞HCT116的增殖抑制活性略强于国外报道的PI3K抑制剂GDC-0941，并且可能作用于mTORC2，我们将在此基础上申请国家自然科学基金进行更深入的研究，验证我们的发现。. 在国家自然科学基金的资助下，课题组已发表8篇SCI论文，还有2篇SCI论文正在写作。课题组参加了3次国内学术会议，发表了1篇会议摘要；申请了2篇中国发明专利，还有1篇发明专利正在写作。共培养4名硕士研究生。.本项目为发现全新结构的具有自主知识产权的NSCLC靶向治疗侯选药物奠定了基础；为EGFR-TKIs获得性耐药NSCLC的精准治疗提供新的治疗方向。

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