Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.
Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.
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DOI：
10.1021/acschembio.2c00334
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发表时间：
2022-09-16
影响因子：
4
通讯作者：
Henderson, Mark J.
中科院分区：
文献类型：
--
作者：
Sanchez, Tino W.;Ronzetti, Michael H.;Owens, Ashley E.;Antony, Maria;Voss, Ty;Wallgren, Eric;Talley, Daniel;Balakrishnan, Krishna;Porello, Sebastian E. Leyes;Rai, Ganesha;Marugan, Juan J.;Michael, Samuel G.;Baljinnyam, Bolormaa;Southall, Noel;Simeonov, Anton;Henderson, Mark J.
在化学探针开发和药物发现过程中，确定分子的作用机制是至关重要的。细胞热漂移分析(CETSA)是确认具有药理作用的小分子在细胞中参与靶点的一个有价值的工具。CETSA直接检测配体和蛋白质靶标之间的生物物理相互作用，这可以改变蛋白质的展开和聚集特性，以响应热挑战。在传统的CETSA实验中，每个温度都需要一个单独的样品，这限制了吞吐量，需要进行实质性的优化。为了从单个样品中获取蛋白质的完整聚集态，我们开发了一个原型实时CETSA(RT-CETSA)平台，将实时聚合酶链式反应(RT-CETSA)与CCD摄像机相结合来检测发光。一种热稳定的纳米乳铁素酶变异体(ThermLuc)经过生物工程处理，可以在高于90°C的温度下展开，并在与不同的目标融合时与监测目标交战事件兼容。利用表征良好的乳酸脱氢酶α抑制剂，RT-CETSA与酶、生物物理和其他基于细胞的检测显示出显著的相关性。为了提高RT-CETSA检测靶标结合的灵敏度，开发了一条数据分析流水线。RT-CETSA技术提高了CETSA方法的能力，并有助于识别细胞中的配体-靶参与，这是评估小分子作用机制的关键步骤。
Determining a molecule’s mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein’s unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered to withstand unfolding at temperatures greater than 90 °C and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.
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影响因子：
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作者：
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