title: Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.
abstract: BACKGROUND
  Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;
  
  
  RESULTS
  We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.
  
  
  CONCLUSIONS
  Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.
authors.affiliation: Burnham Institute for Medical Research, La Jolla, California, USA.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Michael
authors.initials: M
authors.lastname: Cuddy
authors.forename: Vincent Chih-Wen
authors.initials: VC
authors.lastname: Shu
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Yip
authors.forename: Charitha
authors.initials: C
authors.lastname: Madiraju
authors.forename: Paul
authors.initials: P
authors.lastname: Diaz
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Matsuyama
authors.forename: Muneshige
authors.initials: M
authors.lastname: Kaibara
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Taniyama
authors.forename: Stefan
authors.initials: S
authors.lastname: Vasile
authors.forename: Eduard
authors.initials: E
authors.lastname: Sergienko
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D053181:Caspase 8 / Q000378:metabolism; D002626:Chemistry, Pharmaceutical / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D007249:Inflammation; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D011480:Protease Inhibitors / Q000494:pharmacology*; D054852:Small Molecule Libraries; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
chemical_list: D019151:Peptide Library; D011480:Protease Inhibitors; D054852:Small Molecule Libraries; D010447:Peptide Hydrolases; D053181:Caspase 8