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Identification of iNOS Inhibitors Using InteraX^TM

^* To whom correspondence should be addressed. E-mail: philip.mallinder{at}astrazeneca.com.

	Abstract

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^TM enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of -galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in -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 -galactosidase activity. The iNOS InteraX^TM 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 InteraX^TM and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX^TM compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit -galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in -galactosidase activity. Many of these were known inhibitors of iNOS. after IC₅₀ determination in InteraX^TM and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS. (Journal of Biomolecular Screening XXXX:xx-xx)

First published on February 11, 2009, doi:10.1177/1087057109331476

Journal of Biomolecular Screening 2009;14:263.

A more recent version of this article appeared on March 1, 2009


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