Peptide PROTACs: an experimental framework to evaluate genetically encoded targeted degradation in human cells
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Embargo End Date
2025-08-06
ICR Authors
Authors
Portillo Malumbres, M
Document Type
Thesis or Dissertation
Date
2025-02-06
Date Accepted
Abstract
Small molecule PROTACs are emerging as a promising therapeutic strategy for the targeted degradation of disease-related proteins. However, their development remains laborious and unpredictable. To accelerate target selection and provide substantive evidence for prioritising targets in small molecule PROTAC development, this project focuses on the development of genetically encoded peptide PROTACs. The main advantage of using peptide PROTACs is that this approach allows medium-throughput testing of the spatiotemporal compatibility between E3 ligases and their targets, as well as the phenotype of dose-dependent target degradation.
The aim of this work is to design and express genetically encoded peptide PROTACs in human cells to (i) expand the range of E3 ligases that can be recruited by PROTACs and (ii) explore their potential to mediate targeted protein degradation. These peptide PROTACs consist of a degron-derived peptide for E3 ligase recruitment and a SLiM-binding peptide to target specific proteins. To achieve these goals, I have developed a framework that allows fast and cost-effective testing of peptide PROTACs in human cells. This system includes a CRISPR-Cas9 engineered cell line optimised for inducible and dose-responsive protein expression, a functional peptide PROTAC design, and a triple fluorophore reporter to assess their function.
Using this framework, a set of degron-derived peptides were validated for efficient recruitment of endogenous E3 ligases. Expanding the toolkit of E3 ligases opens new possibilities for designing PROTACs that harness E3-specific attributes such as subcellular localisation and cell state-specific activity. Additionally, a proof-of-concept design demonstrated that peptide PROTACs can mediate target protein degradation, offering valuable insights into the spatiotemporal compatibility of E3 ligases and their targets.
Citation
2025
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Short Linear Motif