High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.
High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.
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DOI：
10.1021/acschembio.8b00421
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发表时间：
2018-08-17
影响因子：
4
通讯作者：
Levine B
中科院分区：
文献类型：
--
作者：
Chiang WC;Wei Y;Kuo YC;Wei S;Zhou A;Zou Z;Yehl J;Ranaghan MJ;Skepner A;Bittker JA;Perez JR;Posner BA;Levine B
自噬是一种溶酶体降解途径，在细胞稳态、发育、免疫、肿瘤抑制、代谢、预防神经退行性变和延长寿命中发挥着至关重要的作用。因此，自噬的药理学刺激可能是预防或治疗某些人类疾病和/或衰老的有效方法。我们试图建立一种方法来开发特异性诱导自噬的新化合物。为此，我们开发了两种检测方法来鉴定针对自噬诱导关键调节节点的化合物，特别是 Bcl-2（自噬的负调节因子）与 Beclin 1（Beclin 1/VPS34 脂质激酶复合物的变构调节剂，在自噬启动中发挥作用）的结合。这些测定使用分裂荧光素酶测定来测量细胞中的 Beclin 1/Bcl-2 结合，或使用 AlphaLISA 测定来直接测量体外的直接 Beclin 1/Bcl-2 结合。我们筛选了两个不同的化合物库，其中包含约 300K 化合物，以鉴定破坏 Beclin 1/Bcl-2 结合并诱导自噬的小分子。鉴定出三种新化合物，可直接抑制 Beclin 1/Bcl-2 相互作用，IC50 在微摩尔范围内，并增加自噬通量。这些化合物没有表现出显着的细胞毒性，并且与促凋亡 Bcl-2 家族成员 Bax 和 Bim 的 BH3 结构域相比，它们选择性破坏 Bcl-2 与 Beclin 1 的 BH3 结构域的结合。因此，我们已经确定了候选分子，可作为开发有效且选择性的 Beclin 1/Bcl-2 抑制剂的先导模板，这些抑制剂在临床上可用作自噬诱导剂。
Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction – specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ~300K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared to the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.
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晶体结构和生化分析表明 Beclin 1 是一种新型膜结合蛋白
影响因子：
44.1
作者：
通讯作者：
--
DOI：
10.1073/pnas.1305623110
发表时间：
2013-05-07
影响因子：
11.1
作者：
Decressac, Mickael;Mattsson, Bengt;Bjorklund, Anders
通讯作者：
Bjorklund, Anders
影响因子：
19
作者：
Levine B;Liu R;Dong X;Zhong Q
通讯作者：
Zhong Q
影响因子：
64.8
作者：
He, Congcong;Bassik, Michael C.;Moresi, Viviana;Sun, Kai;Wei, Yongjie;Zou, Zhongju;An, Zhenyi;Loh, Joy;Fisher, Jill;Sun, Qihua;Korsmeyer, Stanley;Packer, Milton;May, Herman I.;Hill, Joseph A.;Virgin, Herbert W.;Gilpin, Christopher;Xiao, Guanghua;Bassel-Duby, Rhonda;Scherer, Philipp E.;Levine, Beth
通讯作者：
Levine, Beth
影响因子：
21.3
作者：
Mizushima, Noboru;Levine, Beth
通讯作者：
Levine, Beth