Enhancing intracellular accumulation and target engagement of PROTACs with reversible covalent chemistry

Abstract

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton’s Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

“…Similarly, Gray et al reported an example of non-cooperative binding between BRD4-dBET23-CRBN, which generated potent degraders (Nowak et al, 2018). To understand this apparent discrepancy, the formulated hypothesis is that the ternary complex formation not only depends on binding affinity rates and positive cooperativity but also on the absolute concentration of the target and the E3 ligase in cells (Guo et al, 2020). Therefore, quantitative western blots to measure their intracellular levels should be done in parallel to kinetic studies.…”

“…In particular, Xue et al generated covalent irreversible BTK degraders that, in contrast to the previous findings (Tinworth et al, 2019), achieved excellent degradation potency in cells (Xue et al, 2020). Moreover, Guo et al sought to measure the efficacy of BTK PROTACs by comparing different warhead chemistry: reversible non-covalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) (Guo et al, 2020). In parallel, another extended work conducted by London's group has been pursued with the same purpose (Ronen et al, 2020).…”

“…Taking advantage of cyano-acrylamide-based reversible covalent inhibitors (Serafimova et al, 2012), they both generated potent covalent BTK degraders, thereby refusing the notion that the sub-stoichiometric MoA is the only prerequisite to produce effective PROTACs. The same authors showed the importance of PROTAC intracellular accumulation and target engagement that is significantly enhanced by the cyano-acrylamide-based reversible covalent chemistry (Guo et al, 2020). To explain the efficacy of covalent PROTACs, it is postulated that their strong binding potency probably compensates for the loss of the substoichiometric mode-of-action.…”

“…Indeed, recent works describing the covalent reversible binding that occurs between electrophilic groups and free cysteines on the cell surface, suggested that non-permeable molecules could benefit from a thio-mediated cellular uptake to enhance their transmembrane penetration (Krishnan et al, 2014;Gasparini et al, 2015;Abegg et al, 2017). In the case of Guo's PROTAC RC-1, the increased in-cell accumulation may be the outcome of a fast and reversible reaction with intracellular glutathione, serving as a trap to enhance PROTAC retention (Guo et al, 2020).…”

Proteolysis targeting chimeras (PROTACs) come of age: entering the third decade of targeted protein degradation

“…PROTACs bearing RC POI ligands have been described for BTK targeting PROTACs. 99,100 Interestingly, the RC BTK PROTACs are not as efficacious as covalent PROTACs, despite the fact that RC PROTACs should retain their catalytic nature since the PROTAC can dissociate. This suggests that ubiquitination and degradation may be on a faster time scale than dissociation, or may even impede dissociation.…”