Proteome-wide identification of on- and off-targets of Bcl-2 inhibitors in native biological systems using affinity-based probes (AfBPs)
Proteome-wide identification of on- and off-targets of Bcl-2 inhibitors in native biological systems using affinity-based probes (AfBPs)
复制标题
使用基于亲和力的探针 (AfBP) 对天然生物系统中 Bcl-2 抑制剂的靶向和脱靶进行蛋白质组鉴定
DOI：
10.1002/cbic.201800380
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发表时间：
2018
期刊：
影响因子：
3.2
通讯作者：
Zhichao Zhang
中科院分区：
文献类型：
--
作者：
Ziqian Wang;Zongwei Guo;Ting Song;Xiaodong Zhang;Nianzhe He;Peng Liu;Peiran Wang;Zhichao Zhang
通过小分子抑制剂选择性抑制Bcl-2家族的蛋白质是药物发现中一种有前途的新方法。然而，关于这些分子如何与其细胞靶点相互作用（开和关）的信息非常有限。我们通过将光交联剂引入Nap-1（小分子Bcl-2抑制剂S1 - 6的荧光衍生物），设计并合成了光反应性和“可点击”的亲和探针（AfBP）-Nap-2和Nap-5。所得的三功能探针Nap-2和Nap-5可以富集、可视化并能够在体外和原位鉴定Bcl-2抑制剂的细胞上靶和脱靶。通过大规模基于细胞的蛋白质组分析和使用Nap-2和Nap-5的下拉/蛋白质印迹（PD/WB），微管蛋白被验证为Bcl-2抑制剂（Nap-1和S1 - 6）的脱靶。由于一些众所周知的Bcl-2抑制剂可以阻断Nap-2对微管蛋白的标记，因此初步证明它是一种含BH 3的蛋白。
Selective inhibition of proteins of the Bcl‐2 family by small‐molecule inhibitors is a promising new approach in drug discovery. However, information about how these molecules interact with their cellular targets (on‐ and off‐) is highly limited. We have designed and synthesized photoreactive and “clickable” affinity‐based probes (AfBPs)—Nap‐2 and Nap‐5—by introducing photo‐crosslinkers onto Nap‐1, a fluorescent derivative of small‐molecule Bcl‐2 inhibitor S1‐6. The resulting trifunctional probes Nap‐2 and Nap‐5 can enrich, visualize, and enable the identification of cellular on‐ and off‐targets of Bcl‐2 inhibitors both in vitro and in situ. Tubulin was validated as an off‐target of Bcl‐2 inhibitors (Nap‐1 and S1‐6) by large‐scale cell‐based proteome profiling and pull‐down/western blotting (PD/WB) with Nap‐2 and Nap‐5. It was preliminarily illustrated to be a BH3‐containing protein because some well‐known Bcl‐2 inhibitors can block the labeling of tubulin by Nap‐2.