Advancing targeted protein degrader discovery by measuring cereblon engagement in cells

Breanna L Zerfas; Fidel Huerta; Hu Liu; Guangyan Du; Nathanael S Gray; Lyn H Jones; Radosław P Nowak

doi:10.1016/bs.mie.2022.08.013

Advancing targeted protein degrader discovery by measuring cereblon engagement in cells

Methods Enzymol. 2023:681:169-188. doi: 10.1016/bs.mie.2022.08.013. Epub 2022 Sep 26.

Authors

Breanna L Zerfas¹, Fidel Huerta², Hu Liu³, Guangyan Du⁴, Nathanael S Gray⁵, Lyn H Jones⁶, Radosław P Nowak⁷

Affiliations

¹ 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. Electronic address: breannal_zerfas@dfci.harvard.edu.
² Center for Protein Degradation, Dana-Farber Cancer Institute, Boston, MA, United States.
³ 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.
⁴ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States.
⁵ Department of Chemical and Systems Biology, ChEM-H, Stanford Cancer Institute, School of Medicine, Stanford University, Stanford, CA, United States.
⁶ 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. Electronic address: lyn_jones@dfci.harvard.edu.
⁷ 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. Electronic address: radoslawp_nowak@dfci.harvard.edu.

PMID: 36764756
DOI: 10.1016/bs.mie.2022.08.013

Abstract

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^CRBN 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.

Keywords: BODIPY™-lenalidomide; Molecular glue; NanoBRET™; Occupancy biomarker; PROTAC; Target engagement; Targeted protein degradation.

MeSH terms

Lenalidomide / pharmacology
Ligands
Proteolysis
Ubiquitin-Protein Ligases* / metabolism
Ubiquitination

Substances

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Ubiquitin-Protein Ligases
Lenalidomide
Ligands