新型FGF2拮抗肽衍生物抗卵巢癌活性与初步机制研究

The cytotoxic drugs such as Cisplatin(DDP) are the most common chemotherapy drugs for ovarian cancer treatment. However, the studies of targeted drugs have not yet been got enough attention in this field. Looking for new targets and targeted drugs is urgently needed. It has been found FGF2 is a potential therapy target in cancer in previous literatures. But all clinical trials about FGF2 antagonists are unsuccessful on account of selectivity, toxicity and other issues. In our previous work, we have confirmed FGF2 is an effective target in ovarian cancer treatment via the analysis of large clinical database, samples and cellular level. Moreover, we found that P7, the FGF2 antagonistic short-peptide which have prominent anti-tumor activity, can not be used as medicine due to short half-life and poor selectivity. Therefore, we modified P7 to get a better stability and selectivity and higher activity novel FGF2 antagonist P25. With the mechanism of P25 targeted FGF2 remaining to be confirmed in follow-on work, we assume this mechanism that different from classical antagonists. Based on the status, FGF proteins are chosen as control group to evaluate the specificity and targeting effects of P25 via SPR and other research methods. Then, ITC, cocrystallization, point mutation, dynamic simulation and so on will be used to clarify molecular mechanisms of P25 targeted FGF2. MTT, colony forming tests, transwell, cell apoprosis, xenograft model anti-tumor assays, PDX model and other methods will be used to assess the anti-cancer activity in vitro and in vivo and chemotherapy increase-sensitivity of P25 for ovarian cancer treatment. This project intends to provide a new strategy for the drug research of FGF2 antagonists and a novel therapeutic target and potential drug in ovarian cancer.

卵巢癌（OC）一线化疗仍为顺铂等细胞毒药物，其靶向药物研发滞后，亟待新靶点和靶向药物。FGF2（F）是OC等癌症治疗的潜在靶点，但因选择性和毒性，其抑制剂药物的临床试验均失败。我们在前期从临床大数据、标本和细胞层面等确证F是OC的治疗有效靶点。也发现具有抗肿瘤活性的F拮抗短肽P7等，但半衰期短或选择性差限制了成药。在前期优化P7等得到稳定、选择性可能极强、有良好体外抗OC活性的F拮抗肽衍生物P25（P）。但其特异靶向F的抗OC活性与机制还有待确证。我们假设P通过与经典拮抗剂不同的机制作用F，具有良好抗OC活性。本项目拟用F家族蛋白等对照，用SPR等确证P的特异靶向选择性；通过ITC、共结晶、点突变、动力学模拟等阐明P靶向F的分子机制；通过MTT、侵袭、凋亡、异种移植瘤和PDX模型等研究P体内外抗OC及增敏化疗的药理药效。本项目将为F拮抗剂药物研发提供新策略，为OC治疗提供新靶点和候选药物。

卵巢癌（OC）一线化疗仍为顺铂等细胞毒药物，其靶向药物研发滞后，亟待新靶点和靶向药物。FGF2（F）是OC等癌症治疗的潜在靶点，但因选择性和毒性，其抑制剂药物的临床试验均失败。我们在前期从临床大数据、标本和细胞层面等确证F是OC的治疗有效靶点。也发现具有抗肿瘤活性的F拮抗短肽P7等，但半衰期短或选择性差限制了成药。在前期优化P7等得到稳定、选择性可能极强、有良好体外抗OC活性的F拮抗肽衍生物P25（P）。通过MTT实验检测F对OC细胞A2780、CP70增殖的影响和P对F诱导的OC细胞CP70增殖的影响；通过流式周期实验检测P对F诱导的CP70细胞周期的影响；通过Western Blot实验检测P对F诱导CP70细胞中FGF2/FGFR下游信号通路（FRS2a及其下游ERK和AKT）活化的影响。并用BALB/c裸鼠建立A2780异位移植瘤的裸鼠荷瘤模型，通过腹腔注射化合物P（60mg/kg/day）给药，记录肿瘤的大小和体积以及裸鼠体重；对B6小鼠一次性给予高剂量的P（1500mg/kg；尾静脉注射），测量2周小鼠的体重大小变化。结果发现F能呈剂量依赖的促进OC细胞A2780和CP70的增殖， P能剂量依赖的抑制F诱导的OC细胞CP70的增殖；流式周期实验表明，P能阻滞F诱导的CP70细胞周期在G2/M期；Western Blot实验表明，P能明显抑制F诱导CP70细胞中p-FRS2a、p-AKT、p-ERK蛋白的表达，且异位移植瘤的裸鼠荷瘤模型表明P能较好的抑制体内肿瘤的增长；急性毒性试验的小鼠体重无明显改变。从而得出靶向F肽类衍生物P具有良好的体内外抗OC活性和生物安全性，为OC治疗提供了新的靶向治疗候选药物。