Center for Protein Degradation, Dana-Farber Cancer Institute, Boston, MA, United States; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States.
Center for Protein Degradation, Dana-Farber Cancer Institute, Boston, MA, United States.
Methods Enzymol. 2023;681:169-188. doi: 10.1016/bs.mie.2022.08.013. Epub 2022 Sep 26.
Measurement of target engagement in cells is critical to understand the molecular pharmacology of drugs and chemical probes. Many targeted protein degraders engage the E3 ligase CRL4 and induce proximity with target neosubstrates resulting in their polyubiquitination and subsequent proteasomal degradation. Here we describe the development of a sensitive and robust cellular NanoBRET-based assay that measures occupancy of the CRBN ligand binding site. The assay is based on a bioluminescence resonance energy transfer (BRET) between NanoLuc luciferase tagged CRBN and a BODIPY-lenalidomide tracer which can be competed out by CRBN ligands, including PROTACs and molecular glues. The assay is compatible with a 384-well plate setup, does not require transfections and can be performed in a single day with only 3-4h of laboratory time. The protocols can be used to design other NanoLuc fusion engagement assays based on BODIPY tracers.
测量细胞内的靶标占有率对于理解药物和化学探针的分子药理学至关重要。许多靶向蛋白降解剂与 E3 连接酶 CRL4 结合，并诱导与靶标新底物的接近，导致它们多聚泛素化，随后被蛋白酶体降解。在这里，我们描述了一种灵敏而稳健的基于细胞 NanoBRET 的测定方法，用于测量 CRBN 配体结合位点的占有率。该测定法基于 NanoLuc 荧光素酶标记的 CRBN 与 BODIPY-来那度胺示踪剂之间的生物发光共振能量转移（BRET），该示踪剂可被包括 PROTAC 和分子胶在内的 CRBN 配体竞争出来。该测定法与 384 孔板设置兼容，不需要转染，并且可以在一天内完成，仅需 3-4 小时的实验室时间。该方案可用于设计基于 BODIPY 示踪剂的其他 NanoLuc 融合结合测定法。
