Assays and technologies for developing proteolysis targeting chimera degraders

Abstract

Targeted protein degradation by small-molecule degraders represents an emerging mode of action in drug discovery. Proteolysis targeting chimeras (PROTACs) are small molecules that can recruit an E3 ligase and a protein of interest (POI) into proximity, leading to induced ubiquitination and degradation of the POI by the proteasome system. To date, the design and optimization of PROTACs remain empirical due to the complicated mechanism of induced protein degradation. Nevertheless, it is increasingly appreciated that profiling step-by-step along the ubiquitin-proteasome degradation pathway using biochemical and biophysical assays are essential in understanding the structure–activity relationship and facilitating the rational design of PROTACs. This review aims to summarize these assays and to discuss the potential of expanding the toolbox with other new techniques.

Applications of Bacterial Degrons and Degraders — Toward Targeted Protein Degradation in Bacteria

“…Tight binding of the chimeras is achieved by a mechanism of cooperative binding which leads to high ternary affinities. Preferably, the affinity of the PROTAC-target or PROTAC-E3 complexes to the third component (the E3 ligase or the target, respectively) should be higher than the separate binary affinities of the PROTAC components to its individual binding partners (to the E3 ligase or the target) alone (Gadd et al, 2017;Liu X. et al, 2020). Linkers, usually made of PEG or alkyls, play an important role in enabling molecules to form this coordinative and permissive complex by keeping them at a distance which helps to reduce steric constraints but at the same time allows efficient ubiquitination by the proximity effect.…”

“…The size of a chimera composed of two different ligands is twice as large as traditional drugs, which affects their pharmacokinetics and can potentially cause absorption issues. Surprisingly, PROTAC permeability is relatively high and can be improved by linker modifications or attaching cell-penetrating peptides (Maple et al, 2019;Jin J. et al, 2020;Liu X. et al, 2020). Ligands that bind to the E3 ligase and to the protein of interest can be either small molecules or peptides.…”

Welcome to volume 13 of Future Medicinal Chemistry

“…Our most read article this year, 'Assays and technologies for developing proteolysis targeting chimera degraders,' discussed the landscape of available assays to evaluate proteolysis targeting chimera substrate degradation [4]. Choosing the right assay for characterizing a proteolysis targeting chimera is essential for medicinal chemists to understand the mechanism of action of a proteolysis targeting chimera molecule, making assay development a key asset to this field.…”

PROTACs are effective in addressing the platelet toxicity associated with BCL-XL inhibitors

“…The polyubiquitinated POI can be recognized by 26S proteasome and eventually broken into small peptides ( Figure 3). Various techniques have been developed to characterize each step of this process [51]. Over 50 proteins have been successfully targeted by this technology, including nuclear receptors, protein kinases, epigenetic regulators, neurodegenerative disease-related proteins, regulatory proteins, anti-apoptotic proteins, virus-related proteins, transcription factors, and even E3 ligases [17,52].…”