Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany.
Methods Mol Biol. 2021;2228:237-252. doi: 10.1007/978-1-0716-1024-4_17.
In order to understand the full mechanism of action of candidate drug molecules, it is critical to thoroughly characterize their interactions with endogenously expressed pharmacological targets and potentially undesired off-targets. Here we describe a chemoproteomics approach that is based on functionalized analogs of the compound of interest to affinity enrich target proteins from cell or tissue extracts. Experiments are designed as competition binding assays where free parental compound is spiked at a range of concentrations into the extracts to compete specific binders off the immobilized compound matrix. Quantification of matrix-bound proteins enables generation of dose-response curves and half-binding concentrations. In addition, the influence of the affinity matrix on the equilibrium is determined in rebinding experiments. TMT10 isobaric mass tags enable analyzing repeat binding and dose-dependent competition samples in a single mass spectrometry analysis run, thus enabling the efficient identification of targets, apparent dissociation constants, and selectivity of small molecules in a single experiment. The workflow is exemplified with the kinase inhibitor sunitinib.
为了全面了解候选药物分子的作用机制，彻底描述它们与内源性表达的药理学靶标和潜在非预期的脱靶的相互作用至关重要。在这里，我们描述了一种基于感兴趣化合物的功能化类似物的化学蛋白质组学方法，该方法可以从细胞或组织提取物中亲和富集靶蛋白。实验设计为竞争结合测定，其中将游离母体化合物以一系列浓度掺入提取物中，以从固定化化合物基质上竞争特异性结合物。基质结合蛋白的定量分析可生成剂量反应曲线和半结合浓度。此外，在再结合实验中确定亲和基质对平衡的影响。TMT10 等压质量标签可在单个质谱分析运行中分析重复结合和剂量依赖性竞争样品，从而能够在单个实验中有效地鉴定靶标、小分子的表观解离常数和选择性。该工作流程通过激酶抑制剂舒尼替尼进行了示例。
