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.

中文翻译：

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二酰基甘油激酶的配体结合态势

二酰基甘油激酶（DGK）是信号转导级联的重要组成部分，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，并且需要研究这些多域脂质激酶的同工型特异性功能的方法。在这里，我们利用基于ATP酰基磷酸酯活性的探针和定量质谱法首次定义了来自所有五种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抑制剂利坦色林的异常结合模式，包括与ATP结合区不​​同的非典型C1结构域的相互作用。不料，利坦色林的解构产生片段化合物，该片段化合物通过保守的结合模式和增强的对激酶组的选择性来阻断DGKα活性。总的来说，我们的研究说明了化学蛋白质组学为基于片段的先导发现分析脂质激酶的蛋白质-小分子相互作用的能力。