Investigation into the Mechanism of Action Behind Small Molecule-Mediated Enhancement of the 20S Proteasome through the Development and Utilization of Novel Nitrogenous Heterocyclic Compounds 20 views

The ubiquitin-independent protein degradation pathway is a crucial aspect of maintaining protein homeostasis in cells, though a variety of factors can lead to its dysregulation. The accumulation and aggregation of intrinsically disordered proteins, or the pathway’s main substrates, is often involved in this dysregulation through direct inhibition of the degradation pathway, which has implications in a variety of neurodegenerative diseases. Enhancement of this pathway by targeting the 20S proteasome, or the enzyme responsible for the protein degradation activity, has been a recently proposed therapeutic target for overcoming this protein-associated dysregulation. Several proof-of-concept studies have led to the discovery and generation of many known small molecules capable of inducing this enhancement. While these advancements have begun demonstrating the therapeutic applicability of this disease intervention strategy, several barriers remain for the field. Such challenges include the obscurity in the mechanism of action for these small molecule enhancers and the continued need for more diverse scaffolds, which would increase both the field’s chemical space and current potency levels. To address these challenges, several classes of heterocyclic compounds were investigated for their potential to impact the activity of this pathway. Select members of the circumdatin natural product family were among the first compounds assessed. While many of these natural products lacked suitable levels of activity, this study completed the first known total synthesis of circumdatin D and identified circumdatin E as a moderate inhibitor of the 20S proteasome in purified conditions. Five additional classes of nitrogenous small molecules were examined for their abilities to enhance 20S proteasome activity. Only the hydroxyquinolinone compounds demonstrated this desired activity, but many members of this novel class went on to increase the degradation of both wt and mutant forms of ɑ-synuclein in purified and cellular conditions. Finally, studies continuing to elucidate the mechanism of action for small molecule enhancers of the 20S proteasome were undertaken using two different approaches. The first strategy evaluated a series of novel molecular staples while the second assessed smaller covalent scaffolds. In both cases, the binding of both compound types was examined from various angles, including competition and inhibition assays, in-gel fluorescence labeling, streptavidin immunoblotting, and cryogenic electron microscopy (cryo-EM) techniques. Although these binding studies are still ongoing, these efforts have yielded the first evidence of a covalent binding event between one of our small molecules and the 20S proteasome as well as the first set of high-resolution cryo-EM structures for our group.

PHD (Doctor of Philosophy)

20S Proteasome; Intrinsically Disordered Proteins; Circumdatins; Organic Synthesis; Heterocycles; Structure-Activity Relationship (SAR)

English

2025-12-02