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Comprehensive profiling of degrader-protein interactions

ABSTRACT: Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue interactomes. We map the targets of 20 molecular glues across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Eric Fischer

LAB HEAD: Eric Fischer

PROVIDER: PXD054860 | Pride | 2025-07-21

REPOSITORIES: Pride

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			Action	DRS
	esf3_01448.raw	Raw
	esf3_01449.raw	Raw
	esf3_01450.raw	Raw
	esf3_01451.raw	Raw
	esf3_01452.raw	Raw

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Comprehensive profiling of degrader-protein interactions

Project description:Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue interactomes. We map the targets of 20 molecular glues across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

2025-07-21 | PXD054862 | Pride

Comprehensive profiling of degrader-protein interactions

Project description:Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of degrader interactomes. We map the targets of 20 molecular glue degraders across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

2025-07-21 | PXD054761 | Pride

Comprehensive profiling of degrader-protein interactions

Project description:Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of degrader interactomes. We map the targets of 20 molecular glue degraders across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

2025-07-21 | PXD054732 | Pride

Comprehensive profiling of degrader-protein interactions

Project description:Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of degrader interactomes. We map the targets of 20 molecular glue degraders across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

2025-07-21 | PXD054764 | Pride

Continuous evolution of compact protein degradation tags regulated by selective cereblon molecular glues

Project description:Small molecule-triggered protein degradation tags (degrons) are powerful tools for biology, biotechnology, and therapeutics development. Commonly used degrons, however, are large protein domains (typically >100 amino acids) that cannot be easily or cleanly installed in endogenous protein-coding genes in most cell types. Although some zinc-finger (ZF) degrons are theoretically small enough for facile endogenous tagging, all are triggered by small molecules with substantial off-target effects, precluding their use as highly specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glues (MG-PACE) and applied it to evolve ZF degrons that engage cereblon (CRBN) only in the presence of orthogonal thalidomide derivatives. MG-PACE evolved a 36-amino acid ZF degron (SD40) that binds CRBN with high affinity in the presence of PT-179, a thalidomide derivative with no detected neosubstrate activity. The evolved degron is small enough to be efficiently inserted into targeted genomic sites in human cells using prime editing. Human proteins tagged with the evolved degron are rapidly and potently degraded by the otherwise-inert PT-179. To overcome the inactivity of IMiD-based degrons in rodents, we separately used MG-PACE to evolve ZF degrons that engage mouse CRBN, enabling induced target protein degradation in mouse cells. High-resolution cryo-electron microscopy structures of SD40 in complex with CRBN/DDB1 bound to PT-179 or pomalidomide reveal mechanistic insights into the evolution and molecular basis of SD40’s activity and specificity. Collectively, our efforts establish a system for the rapid evolution of molecular glue complexes with novel specificities and compact ZF tags that overcome shortcomings associated with existing degrons.

2024-08-09 | PXD044940 | Pride

Continuous evolution of compact protein degradation tags regulated by selective cereblon molecular glues

2024-08-09 | PXD047665 | Pride

Continuous evolution of compact protein degradation tags regulated by selective cereblon molecular glues

2024-08-09 | PXD047593 | Pride

An engineered Cereblon optimized for high throughput screening and molecular glue discovery

Project description:The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target to the E3 ligase receptor Cereblon (CRBN), however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs which retain all features for ternary complex formation, whilst allowing generation of homogenous and cost-efficient expression in E.coli. Extensive profiling of the construct, shows it to be the “best of both worlds” in terms of binding activity and ease of production. We next designed the ‘Enamine focused IMiD library’ and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape, the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues.

2024-12-06 | PXD057393 | Pride

Ubiquitin-specific proximity labeling for the identification of E3 ubiquitin ligase substrates

Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.

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Ubiquitin-specific proximity labeling for the identification of E3 ubiquitin ligase substrates

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