National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, 20850, USA.
Sci Rep. 2018 Jun 21;8(1):9472. doi: 10.1038/s41598-018-27834-y.
Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.
在生理相关的细胞环境中评估药物与其蛋白靶标的相互作用是临床前药物发现领域的主要挑战。细胞热转移分析（CETSA）通过定量测定配体结合后完整细胞中蛋白热稳定性的变化来实现这种评估。在这里，我们提出了一种均相、标准化、无靶点和高通量（384 孔和 1536 孔格式）的 CETSA 平台的开发和验证，该平台使用了一种分裂 NanoLuciferase 方法（SplitLuc CETSA）。该测定法的广泛适用性已通过各种靶标得到证明，并使用一组经过充分表征的抑制剂将其性能与独立的生化和基于细胞的读数进行了比较。此外，我们还研究了该平台作为高通量筛选的初步测定法的实用性。本文介绍的 SplitLuc CETSA 可用于中高通量应用的靶标结合研究。此外，它还为那些不存在表型或其他基于细胞的测定法的靶标提供了快速的测定法开发和筛选平台。
