Development of targeted protein degradation therapeutics

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the I kappa B alpha phosphopeptide that is recognized by the F-box protein beta-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF(beta-TRCP), ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

Jasmonates (JAs) are a family of plant hormones that regulate plant growth, development, and responses to stress. The F-box protein CORONATINE-INSENSITIVE 1 (COI1) mediates JA signaling by promoting hormone-dependent ubiquitination and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of JA perception remains unclear. Here we present structural and pharmacological data to show that the true JA receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone, (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile), with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the JA co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of JA perception and highlight the ability of F-box proteins to evolve as multi-component signaling hubs.

BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt's lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors, resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors.