Reversible-covalent proteolysis targeting chimeras – opening the door for new targets?

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that tether target proteins to E3 ligases and induce the ubiquitination and degradation of their targets. As therapeutic agents, PROTACs are believed to harbor many advantages over traditional inhibitors – they may have higher selectivity, longer duration of action due to the need to resynthesize the target protein, and can degrade a target solely by binding it without direct inhibition.

While covalent inhibitors can have superior activity and pharmacological properties, it has been postulated that covalent PROTACs will be inferior due to the stoichiometric nature of their activity, compared to the catalytic behavior of PROTACs that bind their targets reversibly. We therefore tested whether cyanoacrylamide-based PROTACs, that combine covalent and reversible binding, can function as potent PROTACs, and compared them to non-covalent and irreversible covalent PROTACs. We focused on Bruton’s Tyrosine Kinase (BTK), a prominent kinase in B-cell lymphoma.

We developed a versatile and generic synthetic approach for the synthesis of cyanoacrylamide-based PROTACs and used it to prepare a series of reversible covalent PROTACs targeting BTK. One of the cyanoacrylamides potently degraded BTK in cells with ~100 nM potency. To our knowledge, this is the first active cyanoacrylamide-based reversible covalent PROTAC. Furthermore, we developed additional PROTACs with similar geometry and distinct chemistries, and discovered a highly potent irreversible covalent PROTAC, as well as a non-covalent PROTAC that efficiently degrades BTK at single digit nM concentrations, which are perhaps the most potent BTK PROTACs reported to date. Proteomics studies confirmed that our PROTACs degrade BTK selectively. Our results confirm the potential of covalent PROTACs as tools against proteins that are difficult to target with noncovalent binders.