Autor: |
Mallinder PR; Department of Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, UK. philip.mallinder@astrazeneca.com, Wallace AV, Allenby G |
Jazyk: |
angličtina |
Zdroj: |
Journal of biomolecular screening [J Biomol Screen] 2009 Mar; Vol. 14 (3), pp. 263-72. Date of Electronic Publication: 2009 Feb 11. |
DOI: |
10.1177/1087057109331476 |
Abstrakt: |
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. |
Databáze: |
MEDLINE |
Externí odkaz: |
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