Harvesting the power of the cell’s own protein degradation mechanisms in drug discovery
Posted 21st October 2020 by Joshua Sewell
Constant production of proteins in living cells is subject to strict quality and concentration control. The ubiquitin-protease system (UPS) is one of the systems responsible for degrading misfolded and damaged proteins. Also, short-lived regulatory proteins that control many critical cellular processes, including cell cycle progression, cell proliferation and differentiation, cell signalling and transcription are degraded by UPS.1
The basic component of the UPS system is E3 ligases, ubiquitin and 26S proteasome. In 2001 Craig Crews and his team reported an elegant proof-of-concept study demonstrating that we can hijack this degradation mechanism for targeted degradation of a disease-related protein. This powerful academic application has resulted in an extremely innovative approach to drug discovery.
Bifunctional molecules consisting of a ligand that binds to an E3 ligase, connected by a linker to another ligand that binds to the protein of interest (POI), are often referred to as degraders. This bifunctional molecule brings the E3 ligase near the POI, which results in the polyubiquitination of the POI by the E3 ligase and subsequent proteasomal degradation (Fig. 1).
Bifunctional degraders thus hijack the ubiquitin-proteasome system to achieve the degradation of a disease-related target protein. Importantly, degraders trigger an artificially induced target degradation, by bringing into proximity to two proteins that normally would not interact.
At present, degraders have been successfully employed in the degradation of different types of target proteins related to various diseases, including cancer, viral infection, immune disorders, and neurodegenerative diseases2. Many of the degrader targets were previously considered undruggable2. Additionally, many studies suggest that degrading a protein is better than inhibiting a protein for anticancer activities.
Figure 1: Targeted protein degradation: A bifunctional degrader containing two small molecule-binding ligands joined together by a linker. One of the small molecule ligands is designed to bind POI whilst the other binds with high affinity to an E3 ligase. This leads to the ternary complex formation and consequent polyubiquitination of POI. The degrader then dissociates from the polyubiquinated POI and initiates another cycle. The polyubiquinated POI is than degraded by the proteasome. Legend: POI – protein of interest, E2 – E2 ligase, Ub–ubiquitin.
The successful interaction between the POI and the E3 ligase relies on a degrader molecule that contains ligands with an adequate affinity for the POI and the E3 ligase connected by an appropriate linker (Fig. 1 and Fig. 2). In contrast to classical drug pharmacology, no functional activity is necessary for either ligand. The moderate to high-affinity ligands targeting the protein of interest (POI) and/or the E3 ligases are perhaps best found by direct binding screens, such as fragment-based or DNA-encoded library (DEL) screens.3
One can even combine learnings from parallel DNA-encoded library and fragment screens to discover novel ligands, as exemplified by Wellaway and colleagues who reported the discovery of a promising candidate molecule targeting the bromodomain and extra terminal domain (BET).4
The potential benefits of DEL screening to discover ligands for generating bifunctional degraders hasn’t gone unnoticed.5 Ligands discovered by DEL screening always possess a solvent accessible vector (this vector is the residual linkage connecting the small-molecule to its DNA barcode), and this vector may serve as a chemical handle for the addition of a bifunctional degrader linker.
Also, being a direct binding screen, DEL screens can find ligands for binding pockets with no obvious effect on the POI’s activity. The binding of ligands to such pockets would otherwise be overlooked by traditional biochemical screens. Lastly, DELs provide the ability to rapidly screen a wide chemical diversity of small-molecules, and a published track record of providing potent and novel chemical matter.
Alex Satz is Senior Director of DEL Strategy and Operations, Wuxi Apptec.
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- Zheng, N., & Shabek, N. (2017). Ubiquitin ligases: structure, function, and regulation. Annual review of biochemistry, 86, 129-157.
- Gao, H., Sun, X., & Rao, Y. (2020). PROTAC Technology: Opportunities and Challenges. ACS Medicinal Chemistry Letters, 11(3), 237-240.
- Lucas, X., Van Molle, I., & Ciulli, A. (2018). Surface Probing by Fragment-Based Screening and Computational Methods Identifies Ligandable Pockets on the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase. Journal of medicinal chemistry, 61(16), 7387-7393.
- Wellaway, C. R., et al (2020). Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening. Journal of Medicinal Chemistry, 63(2), 714-746.
- Matyskiela, Mary E., et al. “A novel cereblon modulator recruits GSPT1 to the CRL4 CRBN ubiquitin ligase.” Nature 535.7611 (2016): 252-257.
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Clearly, unraveling the mechanistic pathways involved in targeted protein degradation has opened up new opportunities for drug discovery and development.