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.
Highlights
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
Johnson et al.[27] showed that ibrutinib is >6 folds more potent than its Michael acceptor saturated ibrutinib analog in a kinase inhibition assay for wild-type Bruton’s Tyrosine Kinase (BTK) (IC50 0.72 nM vs 4.9 nM), while both compounds are potent towards BTK C481S mutant (IC50 4.6 nM vs 4.7 nM)
We serendipitously discovered that cyano-acrylamide groups enhance intracellular accumulation of PROTACs
Summary
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. Irreversible covalent inhibitors, such as ibrutinib, have achieved tremendous clinical success based on their strong target affinities and high target occupancies, it was recently reported that PROTACs with irreversible covalent binders to targeted proteins failed to induce efficient protein degradation[15]. Beginning as a basic science exploration to compare how the warhead chemistry of PROTACs with reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders affect protein degradation, we choose Bruton’s tyrosine kinase (BTK) as a model target to test the hypothesis. Ubiquitination surprise, we discover that cyano-acrylamide-based reversible covalent binder to BTK can significantly enhance drug accumulation and target engagement (TE) in cells. Building on this discovery, we develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor. We hope that this study adds another dimension to improve the cellular efficacy of PROTACs
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