抗寄生虫药硝唑尼特的新用途——抗肿瘤作用靶标发现及机制研究

Anti-parasite agent Nitazoxanide and its structural analogued thiazolides have been reported having cancer cell proliferation inhibition and apoptosis inducing effects (IC50 = 6.8 ±1.3 μM), and additionally, they are selectively effective to those fast proliferating cells and can increase sensitivity to the available anti-tumor agents, for example, Cisplatin. Their potency and priority to be developed to novel anti-tumor agent was reported related to Glutathione S-Transferase (GST). Further explanations on mechanisms remained unclear. However, our preliminary data suggested that thiazolides were not able to inhibit the enzymatic activity of recombinant human GST, and this indicated that thiazolides might not work directly on this target. Additionally, our preliminary work has also indicated STAT3 was largely possible a direct target of Nitazoxanide. Aiming at illuminating the undermined target(s), this project on one hand, will verify the effects of thiazolides on GSTs and STAT3 at both molecular and cellular level, while on the other hand, will 'fish out' the real target(s) from the treated cell lysates using small molecular probes which can covalently bind to its target(s) when exposed to UV light. The fished target(s) will be further validated by measuring its (their) relationships with cell proliferation, apoptosis, cell cycle, GSTs and STAT3 etc., and both in vitro and in vivo research results will contribute to repositioning of thiazolides from anti-parasite drugs to novel anti-tumor agents.

以抗寄生虫药硝唑尼特为代表的苯酰胺基噻唑类化合物近年来被报道具有抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡作用(IC50 = 6.8 ±1.3 μM)，该作用对迅速增殖的细胞有选择性，并能够增加肿瘤细胞对于顺铂等抗肿瘤药的敏感性，具有被开发为抗肿瘤药物的潜力，但作用机制尚不清楚。有人报道硝唑尼特的抗肿瘤增殖作用依赖于细胞内谷胱甘肽转移酶的活性。本课题前期研究结果发现，苯酰胺基噻唑类化合物在细胞外并不能直接影响谷胱甘肽转移酶的活性，说明这一靶点有待验证；同时我们还发现STAT3可能是它的直接作用靶点，其抑制肿瘤细胞增殖的作用可能是对多个靶点共同作用的结果。本课题一方面，通过多种化学和生物学手段从分子和细胞水平分别对谷胱甘肽转移酶和STAT3两个靶点进行验证；另一方面设计合成一系列小分子探针，通过光交联反应直接钓取作用靶标，为最终阐明其抗肿瘤作用机制并开发苯酰胺基噻唑类新型抗肿瘤药物奠定基础。

以抗寄生虫药硝唑尼特为代表的苯酰胺基噻唑类化合物近年来被报道具有抗肿瘤作用，不但能单独诱导肿瘤细胞凋亡，还与多种抗肿瘤药物有协同抗肿瘤作用，且对迅速增殖的细胞有选择性，对正常细胞十分安全。但作用机制和靶点一直不明确。本项目通过结构相似性对比及药物联用等方法，发现了NTZ及同类化合物的两个作用靶标——STAT3和20S蛋白酶体。通过多种药理学和化学生物学手段进行了验证，并确认了NTZ可以作用于STAT3的DNA结合域，通过阻碍STAT3与DNA的结合调控下游基因的表达并诱导细胞凋亡，与目前报道较多的作用于SH2结构域的小分子抑制剂相比更具成药性优势，为开发新机制的STAT3抑制剂提供了优良的先导化合物。同时，采用光交联探针和LC-MS/MS发现NTZ可以与20S蛋白酶体催化口袋的S1’-S2’亚位点相作用并抑制蛋白酶体活性，这一位点区别于之前报道的小分子蛋白酶体抑制剂，对研究新型非肽类小分蛋白酶体抑制剂的研究具有指导意义。研究成果明确了硝唑尼特为代表的苯酰胺基噻唑类化合物的具有良好的抗肿瘤药物应用前景，为开发苯酰胺基噻唑类抗肿瘤新药奠定了基础。