Abstract B072: CURE-PROs: Proof-of-mechanism for the first reversible self-assembling targeted protein degraders
Authors: Giardina Sarah F , Valdambrini Elena , Peel Micheal , Bacolod Manny D , Rothenberg Mace L , Lanman Richard B , Warren J David , Barany Francis 
Published: December 2023

Background: First-generation targeted protein degraders (TPDs) known as Proteolysis-Targeting Chimeras (PROTACs) are hetero-bifunctional molecules that simultaneously bind both a target protein and an E3 ubiquitin ligase, marking the target for proteasomal degradation. PROTACS have shown promise in Phase I studies but are limited by their large size, long development timelines, and emergence of resistance. We sought to overcome these limitations with next-generation TPDs known as CURE-PROs. These small molecule monomers self-assemble and dimerize inside the cancer cell in the presence of the target oncoprotein and E3 ligase to form a quaternary complex that facilitates ubiquitination of the target protein. Due to their small size, the individual CURE-PRO monomer ligands have superior oral and tumor cell absorption and can be rapidly developed or modified utilizing a large library of monomers and linker components. We selected BRD4 as our initial oncoprotein target to validate in vitro and in vivo activity of CURE-PROs.

Methods: Twenty-seven CURE-PRO monomers consisting of derivatives of the BRD4 protein ligands, JQ1 and OXT-015, and ligand monomers that bind E3 ligases (Cereblon, VHL, and MDM2) were combined to create 170 unique CURE-PRO dimers. These combinations were tested in cancer cell lines, in the presence/absence of proteasome inhibitors, and BRD4 protein degradation was assessed by Western Blot. CURE-PRO-mediated degradation of BRD4 was also evaluated in nude mice with bilateral human cancer xenografts using three doses over eight hours. A parallel arm using a BRD4 PROTAC (ARV-825) served as a positive control. The tumors were excised 1, 2, 4, 16 and 40 hours after the last dose and protein expression was analyzed by Western Blot.

Results: Cell screening identified 49 CURE-PRO combinations, consisting of different E3 ligase partners and BRD4 ligands, that induced greater than 50% BRD4 degradation. BRD4 degradation occurred only in the presence of both CURE-PRO monomers and was dependent on the pairs forming a reversible dimer. Five CURE-PRO pairs reduced protein levels by more than 95%, with a half-maximal degradation concentrations (DC50) of 30-100 nM. Consistent with a PROTAC mechanism-of-action, CURE-PRO-induced degradation of BRD4 was inhibited by proteasome inhibitors. In the xenograft models, BRD4 protein levels were reduced to ~40% at 2-4 hours after exposure to a CURE-PRO pair, with full recovery by 40 hours.

Conclusions: Several CURE-PRO pairs, using different E3 ligase partners, achieved BRD4 protein degradation in vitro comparable to PROTACs, with mechanism-of-action through the proteasome system. Our lead CURE-PRO pair significantly degraded BRD4 in a cancer xenograft mouse model. This work constitutes the first proof-of-mechanism of reversible self-assembling drug dimers for targeted protein degradation in vitro and in vivo. Future studies are planned to optimize degradation efficacy and identify if resistance emerges.

Citation Format: Sarah F Giardina, Elena Valdambrini, Micheal Peel, Manny D Bacolod, Mace L Rothenberg, Richard B Lanman, J David Warren, Francis Barany. CURE-PROs: Proof-of-mechanism for the first reversible self-assembling targeted protein degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B072.