Neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease, share a common feature: aggregation of intracellular proteins. In eukaryotic cells, the proteasome system is responsible for most of the non-lysosomal protein clearance, and it can degrade proteins regardless of their ubiquitinated state. As cells age, the proteasomal activity declines due to a decreased subunit expression or disassembly. This proteostasis malfunction leads to protein aggregation and, subsequently, diseased states; hence, the discovery and evaluation of small molecules that can restore proteasomal activity levels, and clear aggregation-prone proteins, represents an attractive therapeutic approach. Advances in the field have identified molecules that increase the proteasome-mediated proteolysis; however, no studies have yet accurately identified their binding site and addressed their off-target toxicity. Recently, we identified the FDA-approved drug miconazole (MO) as a stimulator of the 20S proteasome 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 mechanisms of action and interaction network at a proteome-wide scale is necessary. Here, we’ve used a photoaffinity labeling (PAL) approach to map MO’s interaction network. Through structure-activity relationship (SAR) studies, we’ve determined MO’s key features responsible for proteasomal activation and designed a diazirine-based photoreactive probe that covalently binds to MO’s binding partners in cellular environments. Currently, we are working towards an affinity enrichment step followed by tandem mass spectrometry of photo cross-linked targets to identify the biological targets, and we will subsequently validate them by biophysical or genetic methods.

Interactome mapping by photoaffinity labeling of proteasomal activator and FDA-approved drug Miconazole

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