Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA; Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Cell Chem Biol. 2017 Jul 20;24(7):870-880.e5. doi: 10.1016/j.chembiol.2017.06.007. Epub 2017 Jul 14.
Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.
二酰基甘油激酶 (DGK) 是信号转导级联的组成部分，通过脂质信使二酰基甘油的 ATP 依赖性磷酸化来调节细胞生物学。缺乏直接评估天然生物系统中 DGK 活性的方法，需要研究这些多域脂质激酶的同工型特异性功能。在这里，我们利用 ATP 酰基磷酸活性探针和定量质谱首次定义了所有五种 DGK 亚型的代表性成员的 ATP 和小分子结合基序。我们使用化学蛋白质组学来发现 DGKα 抑制剂利坦色林的一种不寻常的结合模式，包括在与 ATP 结合区域不同的非典型 C1 结构域的相互作用。出乎意料的是，利坦色林的解构产生了一个片段化合物，通过保守的结合模式阻断 DGKα 活性，并提高了针对激酶组的选择性。总的来说，我们的研究说明了化学蛋白质组学在基于片段的先导化合物发现中对脂质激酶的蛋白-小分子相互作用进行分析的强大功能。
