Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells.
Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells.
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DOI：
10.1021/ja505517t
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发表时间：
2014-07-30
影响因子：
15
通讯作者：
Cravatt BF
中科院分区：
文献类型：
--
作者：
Kambe T;Correia BE;Niphakis MJ;Cravatt BF
表型筛选为发现干扰细胞生物学过程的小分子提供了一种手段。然而，通过筛选靶点来识别蛋白质和生化途径在技术上仍然具有挑战性。我们最近描述了使用带有光反应基团和潜在亲和柄的小分子作为综合表型筛选和靶标识别的全功能化探针。这种探针的一般效用，或者任何小分子筛选文库，都取决于它们在细胞中蛋白质相互作用的范围，这是一个很大程度上尚未探索的参数。在这里，我们描述了一个由ugi叠氮化物缩合反应制备的约60个成员的全功能化探针文库的合成，以赋予结构多样性，并引入重氮嘧啶和炔的功能，分别用于靶捕获和富集。基于质谱的深入分析揭示了人类细胞中多种探针靶点，包括酶、通道、接头和支架蛋白以及未表征功能的蛋白质。对于这些蛋白质中的许多，配体尚未被描述。大多数探针-蛋白相互作用在整个探针文库中显示出明确的结构-活性关系，并被细胞中的小分子竞争对手阻断。这些发现表明，完全功能化的小分子可以覆盖人类蛋白质组的不同片段，并有望作为细胞生物学的药理学探针。
Phenotypic screening provides a means to discover small molecules that perturb cell biological processes. Discerning the proteins and biochemical pathways targeted by screening hits, however, remains technically challenging. We recently described the use of small molecules bearing photoreactive groups and latent affinity handles as fully functionalized probes for integrated phenotypic screening and target identification. The general utility of such probes, or, for that matter, any small-molecule screening library, depends on the scope of their protein interactions in cells, a parameter that remains largely unexplored. Here, we describe the synthesis of an ∼60-member fully functionalized probe library, prepared from Ugi-azide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively. In-depth mass spectrometry-based analysis revealed a diverse array of probe targets in human cells, including enzymes, channels, adaptor and scaffolding proteins, and proteins of uncharacterized function. For many of these proteins, ligands have not yet been described. Most of the probe–protein interactions showed well-defined structure–activity relationships across the probe library and were blocked by small-molecule competitors in cells. These findings indicate that fully functionalized small molecules canvas diverse segments of the human proteome and hold promise as pharmacological probes of cell biology.