<p>The proteasome is a multi-subunit protease complex responsible for most of the non-lysosomal protein turnover in eukaryotic cells. This degradation process can be conducted dependent or independent of ubiquitination as different isoforms with different substrate preferences coexist in the cell. Proteasomal activity declines during aging due to a decreased expression of proteasome subunits, complex disassembly, and oxidative stress. This malfunction leads to protein accumulation, subsequent aggregation, and ultimately diseased states. Considering the shared feature of aggregation and accumulation of intrinsically disordered proteins (IDPs) in age-related diseases, and the substrate preference of the 20S isoform for misfolded proteins, enhancing the proteolytic activity of the 20S proteasome has arisen as an attractive strategy to minimize the burden associated with this increased protein load. Recently, we identified the FDA-approved drug miconazole (MO) as a stimulator of the 20S isoform and validated its activity profile in biochemical and cell-based assays. Given its FDA-approved drug status, we considered that to successfully repurpose it, information regarding its binding location within the 20S and network of binding partners, as well as its value in protein homeostasis in age-related diseases are necessary. Herein, we (1) conduct SAR studies to determine MO’s key features responsible for proteasomal activation and obtain molecules with enhanced ability to activate the 20S proteasome; next, using the developed SAR model, we (2) design a diazirine-based photoreactive probe that allows for the identification of MO’s binding partners and location within the 20S proteasome. Lastly, we (3) explore the use of MO to restore the activity of impaired proteasomes by Parkinson’s disease-associated toxic oligomers. This work expands upon previous research avenues by using newer approaches to study this enzymatic complex, and describes methods that can be further used to better establish the role of the 20S proteasome in age-related diseases.</p>