特异性直接激活Bak的小分子筛选和衍化研究

Molecular targeting drugs are used more and more widely in cancer therapies today. Diminished apoptosis plays an important role in cancer development and drug resistance. Therefore, many apoptosis related proteins are targeted to develop molecular targeting drugs, for example, Bcl-2 inhibitor venetoclax has been approved recently to treat CLL. Bak activation is an essential step in the mitochondrial apoptosis pathway, while gene deletions or mutations of Bak activators (such as BIM and PUMA) are one of the roles in cancer development and drug resistance. Although quite a few small molecules targeting the apoptosis pathways are in the clinic or in the clinical trials, there are still no small molecules that could directly activate Bak. Our project proposed to obtain Bak interacting molecules by screening a small molecular library using NMR fragment screening method. Derivation will be performed to improve the affinity and selectivity based on the complex structure of Bak and the small molecule, so that the dissociation constant between the final small molecule and Bak will be around 1 uM. The final small molecule will have a high chance to be a direct Bak activator other than a Bak inhibitor, because many BH3 only direct activators have an affinity with Bak at this range. We could always modify the small molecule to get a direct Bak activator. We will also perform in vitro biochemistry studies and in vivo cell studies to confirm the function of the Bak activator. Small-molecules that directly and selectively activate Bak could avoid the problem caused by gene deletions or mutations of Bak activators, and induce cancer cell death through direct Bak activation. Therefore, these small molecules will have good prospects in cancer treatments.

分子靶向药物在肿瘤治疗中应用广泛。细胞凋亡受到抑制在肿瘤形成和耐药过程中起重要作用，因此很多细胞凋亡相关蛋白质是分子靶向药物的重要靶点，其中Bcl2抑制剂venetoclax已经获准上市。Bak的激活是线粒体凋亡的关键步骤，而Bak激活蛋白质的突变和缺失也是肿瘤形成和耐药的重要因素。虽然有很多针对细胞凋亡的分子靶向药物已经应用或在临床试验，目前尚没有直接激活Bak的小分子化合物。本研究拟通过高度自动化核磁共振片段筛选方法去筛选小分子化合物库获得与Bak相互作用的小分子，并通过结构生物学手段提高其与Bak的亲和力，使其与Bak的解离常数达到1 uM量级（已知的Bak直接激活分子与Bak的解离常数都在这个范围）。获得的小分子将通过分子和细胞水平验证是否能直接激活Bak。直接激活Bak的小分子可以避开Bak激活蛋白质的突变和缺失问题，引起肿瘤细胞凋亡，具有成为抗肿瘤药物的前景。

线粒体细胞凋亡通路是由BCL2蛋白质家族控制的。在凋亡信号产生后，直接激活或间接激活BCL2蛋白质家族的两个分子BAK和BAX来诱导细胞凋亡。BAK和BAX激活后，可以导致线粒体外膜穿孔，释放细胞色素c等分子，引起细胞凋亡。直接激活BAK的小分子可以直接诱导细胞凋亡，用于抗肿瘤药物开发。目前尚无有效的特异性激活BAK的小分子药物。本研究拟通过基于NMR的小分子片段化合物筛选及衍化，并结合其它筛选方法获得直接激活BAK的小分子化合物。获得的小分子化合物将通过一系列体外实验及细胞内实验验证其激活BAK的能力，选择性和特异性，以便用于后期药物开发。. 在研究中，我们通过片段筛选获得了7个片段化合物，在此基础上，我们衍化获得了5个具有更高效的体外结合BAK并导致BAK激活的分子。同时我们也通过虚拟筛选一个大的化合物库获得多个与BAK结合的小分子。进一步在细胞内的验证表明，有1个分子可以直接诱导肿瘤细胞THP.1发生凋亡，而另外两个分子可以与抗凋亡分子抑制剂navitoclax和S63845联合使用促进肿瘤细胞Jurkat和MV-4-11凋亡。BAK敲除实验表明，这些分子通过线粒体凋亡通路起作用。后续实验我们在细胞内敲除了多个可以促BAK激活的仅含BH3蛋白分子，建立了用于验证BAK激活的细胞体系，并利用该体系发现了两个新的促BAK激活的仅含BH3蛋白质BMF和HRK，同时发现BMF和HRK结合于BAK的新位点，为BAK激活分子的开发提供新思路。. 有一部分肿瘤细胞中因为缺乏直接激活BAK的分子，包括BIM和PUMA等，这些缺失在肿瘤发生发展和耐药过程中起重要作用。我们筛选衍化获得的直接激活BAK的小分子可以作用于这一类肿瘤，用于抗肿瘤药物前体的研发。

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