Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.
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.
Hayashi
Hideki
H
Burnham Institute for Medical Research, La Jolla, California, USA.
Cuddy
Michael
M
Shu
Vincent Chih-Wen
VC
Yip
Kenneth W
KW
Madiraju
Charitha
C
Diaz
Paul
P
Matsuyama
Toshifumi
T
Kaibara
Muneshige
M
Taniyama
Kohtaro
K
Vasile
Stefan
S
Sergienko
Eduard
E
Reed
John C
JC
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
D019151:Peptide Library; D011480:Protease Inhibitors; D054852:Small Molecule Libraries; D010447:Peptide Hydrolases; D053181:Caspase 8