靶向自噬关键分子LC3的新型小分子化合物的抗肺癌作用及其机制探讨

The incidence and mortality rates of lung cancer ranked number one all over the world as well as in China, therefore, to develop the novel drugs against lung cancer is of tremendous interests. Currently available data indicate that it is promising to develop anticancer drugs based on targeting the key regulatory protein molecules which play important roles in tumor signaling pathways. The latest research found that there was a close relationship between autophagy and the development of lung cancer, especially the autophagy-associated protein LC3 could be used as a target of lung cancer therapy. Recently, based on the crystallography of LC3 protein, we used the computer-aided drug design (CADD) technology to construct a virtual library of small molecule compounds, which, theoretically, could specifically bind to LC3 domain. From this virtual compound library, via virtual high-throughput screening (HTS) methods, we selected 125 candidate compounds, which presumably bind and activate LC3 domain. We then directly tested these candidate compounds based on their effects on LC3 activity and on the inhibition of lung cancer cell proliferation. We obtained 3 highly promising small molecular compounds (named as #407，#521，#570) with different structures that showed desired activities. Therefore, we intend to explore and clarify the molecular mechanism of #407, the most potent one among the 3 candidates. We will study the effects of #407 on the malignant behaviors like lung cancer cell proliferation, invasion, metastasis and autophagy in vitro, and validate its effects on tumor growth and metastasis with in vivo experiments, and explore the effects of #407 on LC3 activity and molecular mechanisms of action against lung cancer both in vitro and in vivo. As far as we know, few small molecular compounds against lung cancer targeting the autophagy-associated key protein LC3 have been reported. Therefore, the successful implementation of this project will provide valuable information on LC3 as a new target for drugs against lung cancer.

肺癌严重威胁人类健康，其发病率和死亡率均居肿瘤第一位，开发新型抗肺癌药物意义重大。基于肿瘤发生发展信号通路的关键分子开发药物最具前景。已有研究证实：自噬与肺癌关系紧密，诱导自噬发生可杀伤肺癌细胞。细胞自噬关键分子LC3可作为肺癌治疗的药物靶点。我们依据LC3 晶体衍射结果，利用计算机辅助药物设计和虚拟高通量筛选技术，从虚拟化合物库中获得125个分子结构, 经进一步筛选, 发现3个化合物（#407，#521，#570）有激活LC3活性及抑制肺癌A549细胞增殖的作用。因此，本项目拟选效果最好的#407化合物为研究对象，通过细胞实验研究其对肺癌细胞增殖、转移、自噬等生物学行为的影响，也将通过动物移植瘤实验研究其对肺癌的抑制作用，并探讨其影响LC3活性的分子机制。目前尚无以LC3为靶点开发小分子抗肺癌药物的报道。本项目的顺利实施将为肺癌防治及LC3作为抗肺癌药物的新靶点提供资料积累。

肺癌严重威胁人类健康，其发病率和死亡率均居肿瘤第一位，开发基于肿瘤发生发展信号通路的关键分子开发新型抗肺癌药物意义重大且最具前景。基于自噬与肺癌关系紧密，诱导自噬发生可杀伤肺癌细胞的前期研究，细胞自噬关键分子LC3可作为肺癌治疗的药物靶点。本项目在前期研究基础上，以能较好激活LC3活性的#407化合物及结构优化体为研究对象，通过体外细胞实验研究发现其可通过增强和激活多个肺癌细胞株的自噬活性，对多个肺癌细胞株增殖、转移等生物学行为有较好的抑制作用；通过体内动物移植瘤模型研究发现，其对实验动物移植瘤模型的肺癌有较好的抑制作用（*p<0.05，**p<0.01）；分子机制研究发现，其可以通过靶向自噬关键分子LC3通过激活肺癌细胞多个自噬相关蛋白Beclin 1、Atg5/Atg12复合物及信号转导通路Akt/mTOR/p70S6K和TSC/MAPK/ MPK激活和增强肺癌自噬活性从而抑制肺癌增殖生长活性。此外，我们研究了氯喹联合苏尼替尼治疗肾细胞癌的相关作用机制，发现苏尼替尼可以诱导肾癌细胞发生自噬，而氯喹可通过抑制由苏尼替尼诱导的肾癌细胞自噬抑制肾癌细胞的增殖及动物移植瘤生长活性，并初步探讨了其相关机制（*p<0.05）；结合我们先前的相关研究结果，我们对前期研究的化合物进行了相关研究，并探讨了其在体外细胞水平和体内动物移植瘤水平抑制肺癌细胞淋巴血管新生、促进肺癌转移及诱导自噬发生的作用机制（**p<0.01）；基于前期我们的相关研究结果，我们还对前期研究的化合物进行了相关研究，探讨了其对在体外细胞水平和体内动物移植瘤水平对肺癌细胞的G2/M期阻滞相关的体内外活性及其发挥抗肺癌作用的可能的分子机制（**p<0.01）。由于目前尚无以LC3为靶点开发小分子抗肺癌药的报道，本项目的顺利实施为肺癌防治及LC3作为研究自噬的理想分子探针及作为抗肺癌药物的新靶点提供了数据资料积累。