Promega Corporation, Madison, WI, United States.
Promega Corporation, Madison, WI, United States.
Methods Enzymol. 2023;681:81-113. doi: 10.1016/bs.mie.2022.08.055. Epub 2022 Dec 12.
The discovery of new PROTAC molecules is dependent on robust and high-throughput assays to measure PROTAC-protein interactions and ternary complex formation. Here we present the optimization and execution of Lumit Immunoassays to measure PROTAC binding and ternary complex formation in a biochemical format. We demonstrate how Lumit can be used to rank order affinities of small molecules and PROTACs to BRD4(BD1, BD2) and how to measure PROTAC-mediated ternary complex formation of BRD4(BD1, BD2) and E3 Ligase VHL. Results from both biochemical assays correlate with live and lytic cell assays, indicating that Lumit Immunoassays can be used as a high-throughput compatible screening methodology to test new small molecules.
新的 PROTAC 分子的发现依赖于强大的高通量测定法来测量 PROTAC-蛋白相互作用和三元复合物的形成。在此，我们介绍了 Lumit 免疫测定法的优化和执行，以在生化格式下测量 PROTAC 结合和三元复合物的形成。我们展示了如何使用 Lumit 对小分子和 PROTAC 对 BRD4（BD1、BD2）的亲和力进行排序，以及如何测量 BRD4（BD1、BD2）和 E3 连接酶 VHL 的 PROTAC 介导的三元复合物的形成。生化测定的结果与活细胞和溶细胞测定相关，表明 Lumit 免疫测定法可用作高通量相容的筛选方法来测试新的小分子。
