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Discovery of a Small Molecule Probe to Rpn-6, an Essential Subunit of the 26S Proteasome.

A considerable number of essential cellular proteins have no catalytic activity and serve instead as structural components to aid in assembling protein complexes. For example, the assembly and function of the 26S proteasome, the major enzymatic complex necessary for ubiquitin-dependent protein degradation, requires a number of essential protein contacts to associate the 19S regulatory particle with the 20S core particle. Previously, small molecule inhibitors to the active sties of the 20S core particle have been developed but activity of the 26S proteasome could also be altered through disrupting its assembly. . We were interested in discovering a small molecule binder to Rpn-6, as it is among several essential proteins that facilitate 26S assembly, that could be used to further our understanding of the association of the 19S regulatory particle with the 20S core particle. Additionally, we were interested in whether a small molecule-Rpn-6 interaction could potentially be cytotoxic to cancer cells that rely heavily on proteasome activity for survival. A workflow to utilize a one-bead-one-compound library and a thermal shift assay was developed to discover such a molecule. TXS-8, our lead hit, was discovered to have low micromolar range binding affinity for Rpn-6 as well as very limited binding to other proteins. Cytotoxicity of TXS-8 was evaluated in several cell lines, revealing increased cytotoxicity to hematological cancers. Discovery of this peptoid binder to Rpn-6 provides the initial evidence that Rpn-6 could be a druggable target to affect protein degradation and serves as a primary scaffold from which to design more potent binders. We suspect that Rpn-6 could have additional essential roles beyond that as the molecular clamp of the proteasome to help hematological cancer cells survive and that TXS-8 can serve as a useful tool to further elucidate is roles.