The advent of directed protein degraders in drug discovery

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.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimaeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase:PROTAC:target species and how this impacts target degradation selectivity remains elusive. We solved the crystal structure of Brd4-degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.