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Updated: Mar 2, 2026
High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
Published on: November 9, 2020
Targeting endogenous proteins for degradation through the affinity-directed protein missile system
Luke J Fulcher1, Luke D Hutchinson1, Thomas J Macartney1
1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, Dundee, UK.
Abstract:
Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.
Insights
Researchers developed an affinity-directed protein missile (AdPROM) system for targeted protein degradation. This novel approach uses polypeptide binders to recruit target proteins, like SHP2 and ASC, for destruction, overcoming limitations of gene knockouts and RNA interference.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Targeted proteolysis offers advantages over CRISPR/Cas9 and RNA interference for protein manipulation.
- Existing methods like gene knockouts are irreversible, and RNA interference can have off-target effects and incomplete knockdown.
- A versatile system for selective protein degradation is needed for research and therapeutics.
Purpose of the Study:
- To develop and validate a novel affinity-directed protein missile (AdPROM) system for targeted degradation of endogenous proteins.
- To demonstrate the efficacy of AdPROM using synthetic monobodies and VHH nanobodies against specific target proteins.
- To investigate the biological consequences of AdPROM-mediated protein depletion.
Main Methods:
- Engineered AdPROM system by tethering the von Hippel-Lindau (VHL) protein to polypeptide binders.
- Utilized synthetic monobodies targeting protein tyrosine phosphatase SHP2.
- Employed a VHH nanobody targeting human ASC protein.
- Assessed degradation of SHP2 and ASC in human cell lines.
- Evaluated the impact of SHP2 depletion on cellular processes.
Main Results:
- Demonstrated highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC proteins in human cell lines.
- Showcased the ability of synthetic polypeptide binders to recruit target proteins to the CUL2-E3 ligase complex.
- Confirmed that AdPROM-mediated loss of SHP2 affects its known biological functions.
- Established the feasibility of using small polypeptide binders for targeted protein destruction.
Conclusions:
- The AdPROM system provides a powerful and adaptable tool for targeted protein degradation.
- Small polypeptide binders can be effectively utilized for selective recruitment and degradation of endogenous proteins.
- AdPROM offers a promising alternative to existing gene modulation techniques with potential therapeutic applications.
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