新型STAT3小分子抑制剂的结构优化和生物学研究

As STAT3 is a master transcriptional regulator and regulates the expression of genes involved in a variety of malignant processes, STAT3 inhibitors are currently under intense development preclinically and clinically in the oncology area. The primary molecular target has so far been the phosphotyrosine-SH2 domain interactions that stabilize active STAT3 dimers. A viable strategy to stop STAT3 dimerization is to design inhibitors blocking its SH2 domain phosphotyrosine binding site. Many of small molecule inhibitors of STAT3 are nonspecific, contain reactive groups, or are not potent in cell-based assays, therefore the search for more druggable STAT3 inhibitors with high potency and excellent bioavailability remains extremely important. By targeting pentapeptide-binding region (binding region of Phe710-Lys709-Thr708- Lys707- Leu706) of STAT3 SH2 domain we gained a novel of small molecule inhibitor of STAT3 Zmm-5e with potential inhibitory activity, better selectivity and better drugguabilty. This project is to carry out the following research work: according to molecular docking results of Zmm-5e with pentapeptide-binding region of STAT3 SH2 domain, the totally structural optimization combined with molecular biological activity results to prove the molecular binding mode with STAT3 target protein, and to improve the inhibitory activity, selectivity and drugguabilty; biological studies to insight into the molecular mechanism of action, to prove that with such a long small molecule occupying the pentapeptide-binding region of SH2 domain-superficial protein-protein binding sites on the protein surface is a new and effective strategy to inhibit STAT3-STAT3 interaction; a preliminary evaluation of druggability of active compounds to identify lead compounds. The research results of this project will not only lay the foundation for small-molecule STAT3 dimerization inhibitors, but also be of an important significance for the design of the small molecule inhibitors for other protein-protein interaction..

STAT3是细胞信号转导和肿瘤发生的核心调节因子，抑制STAT3-STAT3相互作用是肿瘤药物研究的热点方向，占据STAT3 SH2结构域的磷酸酪氨酸结合位点是目前抑制STAT3二聚化小分子抑制剂设计的核心，这类分子由于类药性低影响其深入的研发。我们前期靶向STAT3 SH2新的五肽结合区域，获得了具有显著抑制活性、较好选择性和类药性的STAT3小分子抑制剂Zmm-5e。现将开展如下工作：根据Zmm-5e与五肽结合区域的对接结果，进行系统的结构优化，结合分子水平活性研究结果，确证活性分子与STAT3靶蛋白的作用模式，并提高活性、选择性和类药性；开展生物学研究，阐明分子作用机制，证明用小分子占据SH2的五肽结合区域-表浅的蛋白-蛋白结合位点是抑制STAT3二聚化的一种有效新策略；开展初步成药性评价，确定先导化合物。本项目研究结果对蛋白-蛋白相互作用的小分子抑制剂的设计具有重要的指导意义。

信号传导及转录激活因子3（Signal Transducer and Activator of Transcription 3， STAT3）是细胞质中重要的转录因子，调控信号从细胞外到细胞核的传导。STAT3在多种肿瘤细胞中表达异常并持续激活，与肿瘤的发生和发展密切相关，也与多种激酶抑制剂的耐药相关，抑制STAT3-STAT3 相互作用是肿瘤药物研究的热点方向，占据STAT3 SH2 结构域的磷酸酪氨酸结合位点是目前抑制STAT3 二聚化小分子抑制剂设计的核心，这类分子由于类药性低影响其深入的研发。本项目首次提出使用小分子靶向STAT3 SH2结构域的五肽Phe710-Leu706结合位点干扰STAT3-STAT3相互作用，避免了磷酸基引起的问题，基于前期利用计算机虚拟筛选及结构优化获得苗头化合物Zmm-5e，参照分子于靶蛋白的对接结果，开展了4轮结构优化，获得多个对IL-6/STAT3信号通路抑制活性强的化合物（IC50: 66-200 nM）。表面等离子共振（SPR）和荧光偏振（FP）实验结果验证了化合物与STAT3 SH2结构域的独特结合方式。多个化合物对STAT3高表达的MDA-MB-468细胞和HEL细胞具有较强的增殖抑制作用。Western Blot实验表明，化合物可以明显抑制STAT3中Tyr705、Ser727的磷酸化以及抑制STAT3信号通路下游c-Myc、Mcl-1等基因的表达。为了该类化合物的体内代谢稳定性，开展了3轮的代谢稳定性，确定了该类化合物的代谢位点，其中化合物B66b对STAT3信号通路的IC50为28 nM，是目前已报道的活性最强的STAT3信号通路小分子抑制剂。2019年上半年将完成体内药效学的评价。研究结果对其他蛋白-蛋白相互作用小分子抑制剂的研究具有指导意义。相关研究结果发表SCI研究论文5篇，申请发明专利1项，参加学术会议2次。