Establishment of a suite of assays that support the discovery of proteasome stimulators

Abstract

Background The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing. Methods The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates. Results To demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold. Conclusion Assays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides. General significance A suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.

Small Molecule Modulation of Proteasome Assembly

“…30,35 Herein, we report a new approach that uses small molecules to modulate the dynamic equilibrium between different protea-some complexes, favoring the 20S proteasome, thus mimicking the natural defense response of cells to reduce high levels of IDPs.…”

Small Molecule Enhancement of 20S Proteasome Activity Targets Intrinsically Disordered Proteins

“…Consistent with this model, reduction of the chain length (compound 3 ) or extension of the chain length (compound 4 ) fails to interact efficiently with Arg83 and subsequently resulted in no significant enhancement of 20S-mediated CT-L proteolysis (Figure 3B). Even though compound 2 enhanced 20S proteolysis, its non-drug-like characteristics (negatively charged at physiological pH) and drop in activity at higher concentrations (Figure 3C, >35 μ M, i.e., possible detergent-like behavior at higher concentrations) 26 prompted us to extend our efforts to pursue more physiologically relevant derivatives. Therefore, alkyl chloride 9b was treated with NaI and morpholine to render compound 5 .…”

Activation of the ubiquitin-proteasome system: implications for neurodegeneration, aging, and tumorigenesis

“…In a cellular model (human neuroblastoma cells carrying a mutated SOD1 gene) of Amyotrophic Lateral Sclerosis, PAP1 prevented the aggregation of the mutated SOD1 protein and increased the viability of fibroblasts and neuroblastoma cells upon challenge with the pro-oxidant hydrogen peroxide. Direct activation by small organic compounds was also described 21,22 . Other drugs were described to act on intracellular targets that indirectly modify the catalytic activity of the proteasome, e.g., by promoting or inhibiting post-translational modifications of the proteasome or by inhibiting a deubiquitylase enzyme 23,24 .…”