A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification
A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification
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DOI：
10.1021/jacs.8b07911
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发表时间：
2019-01-09
影响因子：
15
通讯作者：
Marto, Jarrod A.
中科院分区：
文献类型：
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作者：
Browne, Christopher M.;Jiang, Baishan;Marto, Jarrod A.
尽管最近不可逆药物的临床成功，但由脱靶半胱氨酸的不可预测的修饰介导的潜在毒性代表了共价药物计划扩展的主要障碍。了解共价抑制剂的蛋白质组范围的结合概况可以显着加速它们的发展;然而，目前的质谱分析策略通常不提供复杂的选择性抑制剂的共价活性的直接的氨基酸水平读数。在这里，我们报告了CITe-Id的发展，一种新的化学蛋白质组学方法，采用共价药理学抑制剂作为富集试剂与优化的蛋白质组学平台相结合，直接定量剂量依赖性结合在整个蛋白质组的半胱氨酸-硫醇。我们的不可逆CDK抑制剂THZ 1的CITe-Id分析鉴定了几种意想不到的激酶的剂量依赖性共价修饰，包括未充分研究的激酶PKN 3上的先前未注释的半胱氨酸（C840）。这些数据简化了我们开发JZ 128作为PKN 3的新选择性共价抑制剂的过程。使用JZ 128作为探针化合物，我们确定了新的潜在PKN 3底物，从而提供了PKN 3细胞活性的初步分子观点。CITe-Id为当前的化学蛋白质组学平台提供了有力的补充，以表征共价抑制剂的选择性，鉴定新的可寻址半胱氨酸硫醇，并为基于结构的药物设计程序提供信息。
Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.