Maintenance of proteome fidelity is required to preserve the health of an organism in defiance of developmental fluxes, environmental insults, infectious diseases, and the challenges of aging. This crucial role is the responsibility of the proteostasis (protein homeostasis) network, a multicomponent and unified system involving protein synthesis, folding, and degradation. The degradation system is regulated by the proteasome and the autophagy pathways. These proteolytic systems have thus emerged as therapeutic targets for numerous proteostasis disorders. However, their therapeutic regulation is only feasible if there is a fine understanding of how they function, and the mechanisms of crosstalk underlying their cooperative nature. After more than three decades since the discovery of the proteasome, there is still so much to be known about this exquisite enzyme. Proteolysis by the proteasome could be 20S- or 26S-mediated and may or may not be ubiquitin-dependent. The goal of this dissertation was to discover and characterize small molecules that allow for the decoding of 20S-mediated proteolysis and its role in the regulation of autophagy, with the hope of finding new vulnerabilities for proteostasis drug discovery.In this endeavor, two classes of 20S proteasome activators; imidazolines and phenothiazines were identified via high throughput screening (HTS). In a mechanistic study consisting of cellular, biochemical, biophysical and computational approaches, the imidazoline, TCH-165, was found to stabilize the open-gate active conformation of the human 20S proteasome. This translated into enhanced degradation of cancer-driving intrinsically disordered proteins (IDPs) such as c-Myc, and sensitization of both established and primary cancer cells to this molecule. TCH-165 was also found to enhance 20S-mediated clearance of two key SNARE proteins; SNAP29 and STX17, with subsequent inhibition of autolysosome formation. These observations implicate the 20S proteasome as a key regulator of autophagic flux. The clearance of ubiquitinylated proteins was not affected at concentrations required to boost 20S-mediated degradation of these IDPs. Thus, the 20S-ubiquitin-independent pathway could be enhanced without significantly affecting the 26S-ubiquitin-dependent pathway. These findings provide a new targetable vulnerability for IDP-driven cancers and autophagy-associated chemoresistance.In a proof-of-concept approach, the phenothiazine, chlorpromazine was modified to diminish its dopamine D2 receptor (D2R) activity while retaining its ability to enhance 20S-mediated proteolysis of IDPs associated with neurodegenerative disorders. Using these molecules as controls, the AlphaLISA technology was applied in a proteasome-protein degradation system thereby allowing for the development of an assay that allows for the measurement of 20S activation at the cellular and protein levels. These small molecule 20S agonists can therefore serve as leads to further explore the therapeutic potential of 20S activation in proteostasis disorders or as new tools to provide insight into the ambiguous mechanics of 20S-gate regulation and signaling.In an expanded exploration to cover infectious diseases, some imidazolines were found to inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent for tuberculosis (TB). Given the structural differences between human and Mtb proteasome, CRISPRi/dCas9 system was used to validate the Mtb proteasome as a target for this anti-mycobacterium activity.
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