A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity.
Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.
Lead Discovery Department, AGY Therapeutics Inc, South San Francisco, CA 94080, USA.
Anthony M
AM
Smith
John
J
Chan
Donna
D
Oksenberg
Roman
R
Urfer
David S
DS
Wexler
Arnie
A
Ow
Liping
L
Gao
Alanna
A
McAlorum
Shu-Gui
SG
Huang
D000818:Animals; D001414:Bacitracin / Q000494:pharmacology; D001681:Biological Assay; D002384:Catalysis; D002417:Cattle; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D016018:Least-Squares Analysis; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors
D004791:Enzyme Inhibitors; D007328:Insulin; D001414:Bacitracin; D006861:Hydrogen Peroxide; D019704:Protein Disulfide-Isomerases; D004121:Dimethyl Sulfoxide