Abstract
Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.
Keywords:
BiDAC; kinetics; targeted protein degradation; ubiquitination.
MeSH terms
-
Adaptor Proteins, Signal Transducing / genetics
-
Adaptor Proteins, Signal Transducing / metabolism*
-
Biological Assay*
-
Cell Cycle Proteins / chemistry
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / metabolism*
-
Cell-Free System / chemistry
-
Cell-Free System / metabolism
-
HeLa Cells
-
Humans
-
Kinetics
-
Oxindoles / chemical synthesis
-
Oxindoles / pharmacology*
-
Phthalimides / chemical synthesis
-
Phthalimides / pharmacology*
-
Proteasome Endopeptidase Complex / drug effects
-
Protein Binding
-
Protein Domains
-
Protein Processing, Post-Translational*
-
Proteolysis / drug effects
-
Thermodynamics
-
Transcription Factors / chemistry
-
Transcription Factors / genetics
-
Transcription Factors / metabolism*
-
Ubiquitin-Protein Ligases / genetics
-
Ubiquitin-Protein Ligases / metabolism*
-
Ubiquitination / drug effects
Substances
-
Adaptor Proteins, Signal Transducing
-
BRD4 protein, human
-
CRBN protein, human
-
Cell Cycle Proteins
-
Oxindoles
-
Phthalimides
-
Transcription Factors
-
Ubiquitin-Protein Ligases
-
Proteasome Endopeptidase Complex