Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs.

Ronen Gabizon,Yair Herishanu,Paul Gehrtz,Ziv Shulman,Ben-Zion Katz,Liat Avram,Shira Albeck,Tamar Unger,Alexander Brandis,Ella Livnah,Hila Aharoni,Amit Shraga,Neta Gurwicz,Yamit Shorer,Nir London

doi:10.1021/jacs.9b13907

Abstract

Proteolysis targeting chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including substoichiometric degradation of targets. Their scope, though, is still limited to date by the requirement for a sufficiently potent target binder. A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. Using Bruton’s tyrosine kinase (BTK) as a clinically relevant model system, we show efficient degradation by noncovalent, irreversible covalent, and reversible covalent PROTACs, with <10 nM DC50’s and >85% degradation. Our data suggest that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to ibrutinib and exhibit potent degradation of BTK in patient-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs. These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.

Highlights

Proteolysis targeting chimeras (PROTACs) are receiving increasing attention as a new therapeutic modality, as was recently underscored by the first PROTAC, ARV-110, to enter clinical trials.[1]
The motivation for developing reversible covalent PROTACs lies in the combination of the advantages encompassed by covalent binding, such as increased potency and selectivity, while maintaining the reversibility that is considered important for the catalytic nature of PROTAC efficacy
Our results show that both acrylamides and cyanoacrylamides can function as potent and selective PROTACs, including in patient-derived cell lines (Figure 4), with the irreversible IR-2 being among the most potent Bruton’s tyrosine kinase (BTK) PROTACs reported to date

Summary

Introduction

Proteolysis targeting chimeras (PROTACs) are receiving increasing attention as a new therapeutic modality, as was recently underscored by the first PROTAC, ARV-110, to enter clinical trials.[1]. Efficient degradation typically requires high affinity binding to the target as well as optimized linker geometry, to optimize the ternary complex formation. Many targets such as transcription factors,[15,16] protein−protein interfaces,[17,18] or challenging enzyme classes such as GTPases[19] are recalcitrant to ligand discovery. This limits the applicability of PROTACs against such targets. While several covalent PROTACs have been developed and degrade their target successfully,[20−22] there are examples in which the introduction of irreversible binding reduces the potency of PROTACs.[23,24]

Methods

Results

Conclusion