Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics

Area of Science:

• Biochemistry and Molecular Biology
• Pharmacology and Toxicology
• Developmental Biology

Background:

• Targeted protein degradation is a rapidly advancing field utilizing the E3 ligase cereblon (CRBN).
• Immunomodulatory drugs (IMiDs) like thalidomide leverage CRBN for protein degradation, but off-target degradation of CRBN neosubstrates contributes to developmental toxicity.
• Understanding CRBN neosubstrate profiles is crucial for assessing drug safety and efficacy.

Purpose of the Study:

• To develop and apply a targeted assay for quantifying CRBN neosubstrates in vivo.
• To investigate the impact of IMiDs on CRBN neosubstrate levels in rabbit embryo development.
• To establish a method for improved risk assessment of CRBN-binding compounds.

Main Methods:

• Identification of 25 key proteins involved in CRBN-mediated protein homeostasis and embryonic development.
• Development of a targeted assay combining peptide immunoaffinity enrichment and high-resolution mass spectrometry.
• Application of the assay to rabbit embryo samples from pregnant rabbits treated with three IMiDs.

Main Results:

• The assay successfully detected in vivo changes in known and novel CRBN neosubstrates, including SALL4.
• Distinct neosubstrate degradation profiles were observed for thalidomide, lenalidomide, and pomalidomide.
• Degradation patterns varied among the IMiDs, highlighting compound-specific effects.

Conclusions:

• Monitoring a subset of CRBN neosubstrates in developmental systems is recommended for accurate risk assessment of CRBN-binding compounds.
• The developed assay is configurable and adaptable for future research on CRBN biology and drug development.
• This approach enhances the understanding of IMiD-induced developmental toxicity mechanisms.