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Developing novel Targeted Protein Degradation strategies using Directed Evolution

The Challenge

Diseases caused by the accumulation of excess or misfolded proteins or their unintended overactivity, such as Alzheimer's, Parkinson's, Huntington's disease, and certain cancers, pose a significant challenge in medicine. These conditions are often characterised by toxic protein aggregates or chronically active proteins that disrupt normal cellular functions, lead to cell death, and trigger chronic inflammation. Targeted Protein Degradation (TPD) has emerged as an exciting new therapeutic approach to combat these diseases by targeting and eliminating the problematic proteins that drive their progression1. Unlike current TPD strategies, which can inadvertently affect healthy proteins, cause toxicity, or miss their intended targets, we aim to develop a more precise method of TPD that specifically targets disease-associated proteins while minimising off-target effects and toxicity.

The body naturally disposes of damaged proteins through a system known as the Ubiquitin-Proteasome System (UPS)2. In this system, damaged or excess proteins are tagged with ubiquitin, a small protein that signals the cell’s machinery to degrade the tagged proteins. The proteasome, a cellular complex, breaks down these proteins into harmless components. By leveraging this natural process, TPD offers a powerful strategy to selectively eliminate disease-associated proteins, offering the potential for more effective treatments with fewer side effects on healthy tissues.

Our Approach

We are developing a novel approach that exploits the ability of the UPS to more effectively target proteins for degradation. Our strategy focuses on reprogramming a key player of this system, the E3 ubiquitin ligase3, to selectively recognise and degrade harmful proteins associated with disease. Using Phage-Assisted Continuous Evolution (PACE)4, a powerful technique that mimics natural selection in the laboratory, we aim to engineer E3 ligases capable of targeting specific disease-causing proteins. By engineering these ligases to recognise specific substrates, we are working towards a platform that can rapidly produce customised ligases with precise recognition and degradation capabilities, providing a new avenue to treat diseases.

Our Workflow

  1. Identification of reprogrammable E3 ligases and clinically relevant protein targets.
  2. Construct a selection system and test different promoters, linkers, and substrates, among other things.
  3. Achieve E3 ligase activity-dependent phage propagation in the selection system.
  4. Evolution of the E3 ligase towards recognising clinically relevant protein targets using Phage-Assisted Continous Evolution (PACE).
  5. Confirmation of evolved E3 activity in bacterial system and mammalian cell lines.
  6. Further work towards clinical use of the evolved E3 ligase for Targeted Protein Degradation.

Key achievements:

  • We have identified promising E3 ligases and targets.
  • We designed an evolutionary logic to evolve these E3 ligases.
  • We showed that phage propagation depends on the substrate used and the presence of both RNAP subunits.
  • We observed a high background propagation in our system, and we developed possible solutions for the observed background phage propagation.
  • We successfully drifted SIAH1.

We also:

  • Learned the PACE workflow and practiced setting up and running the PACE reactor.

We are currently still working on:

  • Achieving E3 ligase activity-dependent phage propagation in the selection system.
  • Running PANCE and/or PACE to evolve SIAH1/2 towards the recognition of NLRP3.

References

  1. Tsai JM, Nowak RP, Ebert BL, Fischer ES. Targeted protein degradation: from mechanisms to clinic. Nat Rev Mol Cell Biol. 2024;25: 740–757. doi:10.1038/s41580-024-00729-9 ↩

  2. Damgaard RB. The ubiquitin system: from cell signalling to disease biology and new therapeutic opportunities. Cell Death Differ. 2021;28: 423–426. doi:10.1038/s41418-020-00703-w ↩

  3. Cowan AD, Ciulli A. Driving E3 ligase substrate specificity for targeted protein degradation: Lessons from nature and the laboratory. Annu Rev Biochem. 2022;91: 295–319. doi:10.1146/annurev-biochem-032620-104421 ↩

  4. Miller SM, Wang T, Liu DR. Phage-assisted continuous and non-continuous evolution. Nat Protoc. 2020;15: 4101–4127. doi:10.1038/s41596-020-00410-3 ↩