title: Establishment of a suite of assays that support the discovery of proteasome stimulators.
abstract: 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.
authors.affiliation: Departments of Chemistry and Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States.
authors.forename: Darci J
authors.initials: DJ
authors.lastname: Trader
authors.affiliation: Departments of Chemistry and Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States.
authors.forename: Scott
authors.initials: S
authors.lastname: Simanski
authors.affiliation: Departments of Chemistry and Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States.
authors.forename: Paige
authors.initials: P
authors.lastname: Dickson
authors.affiliation: Departments of Chemistry and Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States. Electronic address: kodadek@scripps.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kodadek
mesh_terms: D001095:Arachidonic Acids / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D057888:Iridoid Glucosides; D039823:Iridoids / Q000494:pharmacology; D053978:Pentacyclic Triterpenes; D010455:Peptides / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology; D014315:Triterpenes / Q000494:pharmacology; D000094062:Betulinic Acid
chemical_list: D001095:Arachidonic Acids; D057888:Iridoid Glucosides; D039823:Iridoids; D053978:Pentacyclic Triterpenes; D010455:Peptides; D061988:Proteasome Inhibitors; D011506:Proteins; D054852:Small Molecule Libraries; D014315:Triterpenes; C002769:oleuropein; D046988:Proteasome Endopeptidase Complex; C107594:N-(4-hydroxyphenyl)arachidonylamide; D000094062:Betulinic Acid
keywords: Agonist; High-throughput screening; Mass spectrometry; Parkinson's disease; Proteasome; α-Synuclein