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Journal of Biomolecular Screening
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Identification of iNOS Inhibitors Using InteraXTM

Philip R. Mallinder

Department of Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, UK, philip.mallinder{at}astrazeneca.com

Alan V. Wallace

Department of Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, UK

Gary Allenby

Department of Molecular Biology, AstraZeneca R&D Charnwood, Loughborough, UK

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 InteraXTM enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of β-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in β-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 β-galactosidase activity. The iNOS InteraXTM assay was used to screen ~800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraXTM and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraXTM compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit β-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in β-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraXTM and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS. ( Journal of Biomolecular Screening 2009:263-272)

Key Words: inducible nitric oxide synthase • dimerization • InteraXTM • β-galactosidase • protein-protein interaction

This version was published on March 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 3, 263-272 (2009)
DOI: 10.1177/1087057109331476
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