A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1
A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1
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DOI：
10.1177/2472555217732014
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发表时间：
2018-01-01
期刊：
影响因子：
3.1
通讯作者：
Holbert, Marc A.
中科院分区：
文献类型：
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作者：
McNulty, Dean E.;Bonnette, William G.;Holbert, Marc A.
早期小分子药物发现中的一个持续存在的问题是，使用纯化蛋白质进行初始筛选得到的生物化学效价与随后的疾病相关细胞表型测定中观察到的降低的效价和功效之间经常缺乏等级顺序相关性。细胞热位移试验（CETSA）的引入通过基于配体诱导的蛋白质热稳定性变化直接在活细胞中评估药物靶点接合来弥合这一差距。将CETSA应用于多个药物靶标类别的初步成功促使我们研究用定量的自动化高通量测定法取代低通量的手动密集的Western印迹读出，该测定法将提供足够的能力来使用CETSA作为主要命中鉴定策略。我们介绍了一种高通量的剂量反应细胞热漂移试验（HTDR-CETSA），一个单锅均匀的高密度微量滴定板格式的测定。该检测方法的特点是在HeLa悬浮细胞中融合至DiscoverX 42氨基酸ePL标签的全长靶蛋白的可滴定BacMam表达，促进了基于酶片段互补的配体稳定的可溶性蛋白的荧光定量。该简化格式可用于测定数百种单个化合物/分析员/天的全剂量CETSA曲线。HTDR-CETSA数据生成的底物位点和替代结合模式的抑制剂组蛋白-赖氨酸N-甲基转移酶SMYD 3在HeLa悬浮细胞中的表现出良好的相关性与细胞的机械分析和直接翻译到目标的参与内源性Smyd 3在癌症相关的细胞系中观察到的排序效力。我们设想这种工作流程一般适用于HTDR-CETSA筛选跨越各种各样的可溶性细胞内蛋白质的目标类别。
A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.