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

• Published in: Biochimica et biophysica acta Vol. 1861; no. 4; pp. 892 - 899
• Main Authors: Trader, Darci J., Simanski, Scott, Dickson, Paige, Kodadek, Thomas
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2017
• Subjects: Agonist; agonists; Arachidonic Acids - pharmacology; betulinic acid; Biological Assay - methods; cell culture; cytotoxicity; High-throughput screening; Humans; hydrolysis; Hydrolysis - drug effects; Iridoids - pharmacology; Mass spectrometry; oleuropein; Parkinson's disease; peptides; Peptides - antagonists & inhibitors; Proteasome; proteasome endopeptidase complex; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - metabolism; Proteasome Inhibitors - chemistry; Proteasome Inhibitors - pharmacology; proteins; Proteins - metabolism; proteolysis; Proteolysis - drug effects; Small Molecule Libraries - pharmacology; Triterpenes - pharmacology; α-Synuclein; High-throughput screening; α-Synuclein; Agonist; Parkinson's disease; Mass spectrometry; Proteasome
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2017.01.003

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. 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. 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. 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. 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. [Display omitted] •A workflow is described that allow bona fide proteasome agonists to be identified.•Two compounds from the NIH Clinical collection, AM-404 and MK-866 are shown to be weak, but bona fide proteasome agonists.