PROteolysis TArgeting Chimera (PROTAC): potency, selectivity, and physico-chemical properties

Background and importance: PROTACs are heterobifunctional molecules consisting of two ligands connected by a chemical linker. An “anchor” ligand binds to the substrate binding domain (SBD) of an E3 ubiquitin ligase, and a “warhead” ligand binds to a particular protein of interest (POI) to be targeted. Through binding to both proteins in cells, the PROTAC recruits the POI to a ternary complex (TC) with the E3 ligase. The E3 ligase itself is in complex with an activated ubiquitin (Ub)-loaded E2 ligase, and the TC formation brings the ensemble into proximity of the POI. This induces the (poly)-ubiquitination of the POI at lysine residues, marking it for degradation by the proteasome. Hence, PROTACs act as adapter molecules between the E3 ligase and any chosen POI, hijacking the activity of the cell’s natural protein degradation machinery, i.e. the ubiquitin-proteasome system (UPS).

Targeted protein degradation by PROTACs has emerged as a new modality for the knock down of a range of proteins, with the first agents now reaching clinical evaluation. The degradative mechanism of action of PROTACs sits in stark contrast to traditional small-molecule inhibitors, which typically inhibit targets through binding to a functional or allosteric site, and this presents several notable advantages. Instead of an occupancy driven effect, PROTACs exert their inhibitory effects via “event-driven” pharmacology. This mechanism is catalytic, and PROTAC molecules freed from the TC can elicit degradation of multiple POIs. Crucially, the high catalytic turnover and irreversible action of the UPS allows PROTACs to be used at extremely low concentrations (down to pM) in cells, which represents a major advantage compared to “occupancy-based” inhibitors.

Another key feature of PROTACs is that the binding site/mode of the warhead to the POI is not of primary importance for successful ubiquitination and does not necessarily need to be functional, as long as the warhead provides sufficient affinity to recruit the POI to the complex. This could provide a means to target the estimated 80% of the human proteome thought to be intractable to conventional small-molecule methods (such as protein-protein interactions (PPIs) and scaffolding proteins) due to their lack of a well-defined functional binding site. This strategy to reduce cellular protein levels has generated huge interest and excitement, and at present, there is significant research aimed at understanding the factors that determine functional efficiency.


Top: general structure of a PROTAC. The E3 ligase targeting “anchor” (blue) is connected to the specific POI targeting warhead (green) via a variable linker; Notable examples of anchors/linkers/warheads from the literature are shown Bottom: Mechanism of PROTAC-mediated target degradation via RING-type E3 ligases. (i) Ub transfer from E1 to E2 by trans-thioesterification is followed by complex formation with an E3 ligase; (ii) The PROTAC binds to both the E3 ligase and POI to form a ternary complex, where the E3 ligase is shown as an assembly of scaffolding proteins (Sc), adapter proteins (Ad), and a substrate binding domain (SBD). This brings the E2 ligase into proximity to the POI; (iii) This leads to the transfer of multiple ubiquitin (Ub) units to surface exposed lysine residues; (iv) The resulting polyubiquitin chain is recognised by the proteasome, leading to the proteolytic degradation of the POI; (v) The PROTAC is released and can catalyse the transfer of Ub to additional POIs.

Research: We are researching novel design and synthetic strategies for the modulation of physico-chemical properties of PROTACs. Through SAR studies, we aim to i/ better understand how the molecular properties (e.g. conformation, lipophilicity, solubility,…) of PROTACs and their individual building blocks (anchor/linker/warhead) influence their overall bio-availability/activity, ii/ shed further light on the mostly elusive molecular interactions mediating cooperativity and productivity in ternary complexes, and explore whether these can be modulated through conformational control at the level of the PROTAC itself. This in turn may perhaps inform structural studies of their architectures and dynamics, and iii/ can this knowledge allow achieving POI isoform targeting specificity across families of POIs having structurally related binding sites. On the longer term, we aim to exploit the knowledge gained from these fundamental studies to derive general design criteria/strategies for optimal efficacy, and fast-track the discovery of PROTAC candidate therapeutics for clinical evaluation.