小分子RL71在新位点结合并选择性抑制SERCA2的化学基础及对结直肠癌的治疗学意义

Our previous study found a small molecule RL71 with potential anti-colon cancer activity after a cell-based screen using hundreds of natural products and synthetic organic molecules. The preliminary identification of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 is a cellular target of RL71. More interestingly, RL71 possibly features special binding to SERCA2 at a novel site and inhibits its activity. In the present study, we will confirm the binding of RL71 to SERCA2 by a series of approaches such as affinity chromatography, co-immunoprecipitation, Dot blot and SPR. Furthermore, we will examine the structural properties of RL71 specially binding to SERCA2 including the binding sites and conformation changes, analyze the structure-activity relationship and determine the selectivity of the binding to the different SERCA isoforms by using computer docking, amino acid mutations and circular dichroism spectra. Correspondingly, we will test the biologic events triggered by RL71 binding to SERCA2 at a novel site in human colon cancer cells, including effects on SERCA2 activity, calcium signaling, cell proliferation, differentiation, death and the related cellular signaling pathways. Finally, we will investigate the therapeutic role of selective SERCA2 inhibition in animal models and clinic samples of colorectal cancer and highlight a novel strategy of isoform-specific SERCA inhibition on colorectal cancer therapy.

前期研究筛选得到抗结肠癌活性小分子RL71，初步证实其靶蛋白是钙ATP酶SERCA2，且RL71可能在新位点结合并选择性抑制SERCA2活性。本项目拟通过亲和纯化共沉淀、Dot Blot、SPR等方法确证RL71与SERCA2的结合。进一步运用计算机模拟、关键氨基酸点突变、圆二色谱等方法阐明二者选择性相互结合的结构基础（包括结合位点和结合后引发的构象变化），构效关系以及对不同SERCA亚类的选择强度，揭示SERCA2的新结合位点与选择性酶活抑制的相互关系。在此基础上探讨RL71作用于新位点对人结肠癌细胞产生的生物学效应，包括对SERCA2酶活性和下游钙离子浓度调控信号的影响；对细胞增殖、分化、迁移以及死亡方式的影响；对SERCA2抑制相关的多条信号通路的影响。最后在结直肠癌小鼠模型和临床样本上探讨RL71作用于新位点产生的治疗学效应，为研制靶向治疗结直肠癌的新型SERCA2抑制剂奠定基础。

本研究我们首先通过合成RL71-biotin进行蛋白垂钓和质谱鉴定，并运用Western Blot、免疫荧光、计算机模拟和基因突变等多种方法确证RL71的靶蛋白是SERCA2，并确定二者结合的新位点位于SERCA2膜内侧连接螺旋M7和M8的loop环L78与连接M3和M4的loop环之间的缝隙，是一个崭新的SERCA2结合位点。并且通过比较一系列RL71结构类似物，考察SERCA2蛋白与这类小分子相互作用的构效关系，预测RL71与SERCA2蛋白选择性结合的分子基础。接下来运用细胞模型和分子生物学技术阐明RL71通过结合新位点产生抗结直肠癌作用的分子机制。然后借助动物模型探讨了RL71作用于新位点对结直肠癌的治疗学意义。此外，在课题实施过程中我们还发现RL71通过靶向SERCA2诱导三阴性乳腺癌发生自噬性死亡，扩展了RL71抗肿瘤作用的治疗范围。更值得一提的是，借助靶点明确的小分子RL71我们揭示新的可供药物调控的细胞自噬途径，有望成为肿瘤治疗的新策略。