Identification of iNOS inhibitors using InteraX.
Inducible nitric oxide synthase (iNOS) is active as a homodimer. A cell-based assay suitable for high-throughput screening (HTS) was generated to identify inhibitors of iNOS dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of beta-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in beta-galactosidase activity. Surprisingly, the assay was also able to identify compounds that have the same profile as known inhibitors of fully formed dimeric iNOS by causing an increase in beta-galactosidase activity. The iNOS InteraX assay was used to screen approximately 800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraX and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit beta-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in beta-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraX and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS.
Mallinder
Philip R
PR
Department of Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, UK. philip.mallinder@astrazeneca.com
Wallace
Alan V
AV
Allenby
Gary
G
D000818:Animals; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D019281:Dimerization; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000166:cytology; D051379:Mice; D008954:Models, Biological; D015394:Molecular Structure; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors*; D010078:Oxazines / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014966:Xanthenes / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010078:Oxazines; D011506:Proteins; D014966:Xanthenes; C005843:resazurin; D052247:Nitric Oxide Synthase Type II; D001616:beta-Galactosidase