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High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
Published on: November 9, 2020
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A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation
Ananya A Basu1,2, Chenlu Zhang1, Isabella A Riha1
1Department of Chemistry, Northwestern University, Evanston, IL, USA.
Nature Chemical Biology
|July 4, 2024
Summary
Researchers identified a new E3 ligase, F-box protein 22 (FBXO22), to enhance targeted protein degradation (TPD) using proteolysis-targeting chimeras (PROTACs). This discovery expands the toolkit for degrading disease-related proteins.
Area of Science:
- Chemical Biology
- Molecular Biology
- Genetics
Background:
- Targeted protein degradation (TPD) is a powerful method for eliminating specific proteins.
- Expanding the repertoire of E3 ligases is crucial for advancing TPD applications.
- Existing E3 ligases limit the scope of TPD strategies.
Purpose of the Study:
- To identify novel human E3 ligases capable of facilitating heterobifunctional compound-mediated degradation.
- To discover new E3 ligases for proteolysis-targeting chimera (PROTAC) development.
- To validate the utility of identified E3 ligases in degrading various target proteins.
Main Methods:
- A clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen was employed to identify functional E3 ligases.
- A candidate PROTAC, 22-SLF, was designed and tested for its ability to induce target degradation.
- Mechanistic studies investigated the interaction between the PROTAC and the identified E3 ligase.
- The efficacy of FBXO22-based PROTACs was demonstrated against additional endogenous proteins.
Main Results:
- A CRISPR screen identified F-box protein 22 (FBXO22) as a novel E3 ligase for TPD.
- The PROTAC 22-SLF was shown to induce degradation of FK506-binding protein 12 (FKBP12) via FBXO22.
- 22-SLF was found to interact with cysteine residues C227 and/or C228 in FBXO22.
- FBXO22-based PROTACs successfully degraded bromodomain-containing protein 4 (BRD4) and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein.
Conclusions:
- FBXO22 is a newly identified E3 ligase that can be leveraged for targeted protein degradation.
- FBXO22-based PROTACs offer a versatile platform for degrading diverse endogenous proteins.
- This work expands the E3 ligase toolbox for developing novel TPD therapeutics.
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