新型STAT3信号通路抑制剂KT53504构效关系和抗肿瘤分子机制研究

Signal transducer and activator of transcription 3 (STAT3) is an oncogene constitutively activated in many human cancers. The persistantly activated STAT3 signaling pathway contributes to carcinogenesis, metastasis and chemoresistance.Inhibition of STAT3 signaling pathway has been demonstrated to increase the apoptotic rate of cancer cells and cancer stem cells, STAT3 pathway has been validated as a novel anticancer target. Thus, the identification of small molecules that inhibit STAT3 signaling pathway could be of great therapeutic importance. We previously demonstrated that KT53504，isolated from Polygonum cuspidatum Sieb. et Zucc., is a novel small-molecule STAT3 signaling inhibitor. KT53504 exhibits potent inhibitory effect on IL-6-induced and constitutive activation of STAT3, and significantly inhibits human cancer cells proliferation, especially those with constitutively activated STAT3.However,structure-activity relationships (SAR) and pharmacophores of KT53504 are not yet fully clarified,its target proteins and molecular mechanisms remain unknown.In this project,structural modifications of KT53504 will be first performed to study SAR and identify its relevant pharmacophores;Targeting cell signaling and apoptotic pathways by KT53504 in HepG2 cells will be further investigated; Next, we attempt to identify the target protein(s) of KT53504, a biotinylated affinity analog will be synthesized for use as a probe, which will allow us to isolate and identify the direct target protein of KT53504 by affinity chromatography and by LC-MS/MS technique.According to target protein,molecular docking,molecular mechanisms and physical-chemical properties of KT53504,lead optimisation will be performed to obtain small-molecule inhibitors that selectively target STAT3 signaling pathway; Moreover, STAT3 inhibitor will be used as chemical probe to investigate how the STAT3 signaling pathway regulates self-renewal, apoptosis and differentiation of human colon cancer stem cells; Finally, effects of STAT3 inhibitor on tumor growth will be examined using a model of nude mice xenogragfted with human colon cancer stem cells. The principal goal of this proposal is to develop novel promising small-molecule STAT3 inhibitors that selectively target cancer cells and cancer stem cells with constitutively activated STAT3.

STAT3信号通路是调控细胞生长的一个重要信号通路，和肿瘤密切相关。我们前期研究证实源于虎杖的KT53504是一种新型较强药理活性STAT3信号通路抑制剂((IC50= 2.13μM))，但其构效关系，直接靶蛋白和分子机制尚未清楚。本课题拟以KT53504为先导物,研究构效关系，明确药效团，阐明抑制STAT3信号和诱导肿瘤细胞凋亡的分子机制；同时以KT53504为母体设计合成生物素标记探针，用亲和色谱和LC-MS/MS技术鉴定其直接作用靶蛋白，解析作用位点氨基酸；据靶蛋白结构，分子对接和理化性质，优化先导结构，获得更强活性选择性STAT3抑制剂；在此基础上以抑制剂为化学探针探索STAT3信号对人结肠癌干细胞CCSCs增殖，凋亡和分化的特异性调控效应，评价对CCSCs裸鼠移植瘤的药效。同时将获得1-2个有开发前景的可药性STAT3抑制剂，为特异靶向STAT3信号的新型抗癌药物研发奠定基础。

肿瘤（干）细胞内持续激活STAT3信号从而导致肿瘤起始发生，转移和化疗药抗药性，STAT3已确认为靶向肿瘤(干)细胞精准治疗新靶标。课题组前期研究证实源于治疗癥瘕传统药物虎杖的天然产物KT53504是一种较强药理活性新型STAT3信号通路抑制剂，但其构效关系，直接靶蛋白和分子机制尚未清楚。本课题首先以KT53504为先导物,通过结构修饰研究了其构效关系，阐明了其主要药效基团；然后根据计算机分子对接和理化性质，优化了分子结构，获得了一种更强药理活性的STAT3抑制剂WG81605(IC50=920 nM),达到纳摩尔水平；然后综合利用设计合成生物素标记探针技术和蛋白质亲和色谱技术分离鉴定了先导物的直接作用靶标蛋白是JAK2和IKK-a蛋白，解析了先导物抑制STAT3信号通路的分子机制，在分子水平诠释了其抗肿瘤分子机理。机制研究表明先导物α，β-不饱和酮基团能以Michael addition反应共价修饰靶标蛋白JAK2和IKK-a, 双重抑制STAT3和NFkB转录因子信号通路，从而抑制肿瘤细胞增殖和肿瘤干细胞自我更新，诱导肿瘤（干）细胞凋亡；我们进一步利用人肝脏肿瘤干细胞（Liver Cancer Stem Cells,LCSCs） 研究模型，发现先导物作为STAT3抑制剂化学探针在体外能显著抑制 LCSCs增值，自我更新和肿瘤球形成，并呈剂量依赖性诱导LCSCs细胞凋亡，细胞阻滞在G2/M期。另外，我们利用分子对接和分子模式技术发现KT53504还能与STAT3蛋白SH2结构域上残基的骨架原子形成氢键，能与Lys591和Ser636形成氢键，还可与SH2结构域上的Try705残基形成σ-作用。本课题研究首次证实了先导物KT53504的直接作用靶标是JAK2和IKK-a蛋白，通过双重靶向STAT3和NFkB信号通路同时杀灭肿瘤细胞和肿瘤干细胞，是一种肿瘤干性（cancer stemness）转录因子（STAT3和NFkB）双重抑制剂，本研究也获得了一个具有明显开发前景的类药性STAT3抑制剂WG81605，为靶向肿瘤干性小分子药物研发奠定科学基础，在肿瘤靶向干预治疗中具有应用前景。