Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs

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 DC 50 '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.

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.…”

Proteolysis targeting chimeras (PROTACs) are emerging therapeutics for hematologic malignancies

“…More importantly, P13I more effectively reduced the viability of BTK-dependent wild-type and C418S BTK-expressing HBL-1 DLBCL cells compared to ibrutinib [117]. Subsequently, several potent BTK PROTACs aimed at targeting mutant BTK in ibrutinib-resistant B cell malignancies with improved cellular activities and possessing in vivo potencies were reported by different groups [7,44,[118][119][120]. Recently, a light-controllable PROTAC against BTK, named pc-PROTAC3, was reported to efficiently degrade BTK in Ramos Burkitt's lymphoma (BL) cells upon light exposure.…”

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

“…Photoactivated protac BRD2/3 and Anaplastic lymphoma kinase (ALK) CRBN Oncology, precision medicine [102] Photoactivated protac BRD4 VHL Oncology, prescision medicine [103] Covalent protac BTK and BLK CRBN or VHL Oncology [106] Covalent protac ERRα VHL Metabolic disorders: Type II diabetes. Oncology: Her2+ and triple-negative breast tumors [111] Covalent protac BTK and BLK CRBN Oncology: chronic lymphocytic leukemia [112] Covalent protac Platform for Halo-tagged proteins. VHL Research use [115] Covalent protac Platform for GFP-tagged proteins VHL Research use [117] Click protac BRD4 and ERK1/2 CRBN Oncology [119] Molecular glue RBM39 E3 ligase receptor DCAF15 Oncology [122] Molecular glue RBM39, cyclinK CRBN Oncology [123] Molecular glue DDB1-CDK12 molecular glue CRBN Oncology [124] Molecular glue CDK12-cyclin K CRBN Oncology [125] Allosteric modulator Aspartate decarboxylase PanD E3-independent Research use, anti-microbial agents [131] Ubiquitin-independent degrader Androgen receptor (AR) (F876L) ND (indirectly, CHIP) Oncology: prostate cancer [137] Ubiquitin-independent degrader Her3 (ErbB3)…”

“…One of the members of the series (protac 1, Figure 5 A) was able to degrade the ERR α protein by more than 80% at a low 30 nM concentration [ 111 ]. A very interesting work is the comparative study carried out with a series of non-covalent (NC), reversible-covalent (RC), and irreversible (IR) protacs against BTK, all of which were derived from the BTK binder ibrutinib [ 112 ] ( Figure 5 ). The most potent reversible covalent protac (RC-3) exhibited enhanced selectivity toward BTK compared to the non-covalent (NC-1) and the irreversible covalent (IR-2) protacs used in the study.…”