Primed for Interactions: Investigating the Primed Substrate Channel of the Proteasome for Improved Molecular Engagement.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

ByeTAC: Bypassing an E3 Ligase for Targeted Protein Degradation.

Targeted protein degradation utilizing a bifunctional molecule to initiate ubiquitination and subsequent degradation by the 26S proteasome has been shown to be a powerful therapeutic intervention. Many bifunctional molecules, including covalent and non-covalent ligands to proteins of interest, have been developed. The traditional target protein degradation methodology targets the protein of interest in both healthy and diseased cell populations, and a therapeutic window is obtained based on the overexpression of the targeted protein. We report here a series of bifunctional degraders that do not rely on interacting with an E3 ligase, but rather a 26S proteasome subunit, which we have named ByeTACs: Bypassing E3 Targeting Chimeras. Rpn-13 is a non-essential ubiquitin receptor for the 26S proteasome. Cells under significant stress or require significant ubiquitin-dependent degradation of proteins for survival, incorporate Rpn-13 in the 26S to increase protein degradation rates. The targeted protein degraders reported here are bifunctional molecules that include a ligand to Rpn-13 and BRD4, the protein of interest we wish to degrade. We synthesized a suite of degraders with varying PEG chain lengths and showed that bifunctional molecules that incorporate a Rpn-13 binder (TCL1) and a BRD4 binder (JQ1) with a PEG linker of 3 or 4 units are the most effective to induce BRD4 degradation. We also demonstrate that our new targeted protein degraders are dependent upon proteasome activity and Rpn-13 expression levels. This establishes a new mechanism of action for our ByeTACs that can be employed for the targeted degradation of a wide variety of protein substrates.

Discovery of a non-covalent ligand for Rpn-13, a therapeutic target for hematological cancers.

The ubiquitin-proteasome system serves as the major proteolytic degradation pathway in eukaryotic cells. Many inhibitors that covalently bind to the proteasome's active sites have been developed for hematological cancers, but resistance can arise in patients. To overcome limitations of active-site proteasome inhibitors, we and others have focused on developing ligands that target subunits on the 19S regulatory particle (19S RP). One such 19S RP subunit, Rpn-13, is a ubiquitin receptor required for hematological cancers to rapidly degrade proteins to avoid apoptosis. Reported Rpn-13 inhibitors covalently bind to the Rpn-13's Pru domain and have been effective anti-hematological cancer agents. Here, we describe the discovery of TCL-1, a non-covalent binder to the Pru domain. Optimization of TCL-1's carboxylate group to an ester increases its cytotoxicity in hematological cancer cell lines. Altogether, our data provides a new scaffold for future medicinal chemistry optimization to target Rpn-13 therapeutically.