A novel cyclooxygenase-2-based thin-layer chromatography (TLC) assay to accelerate the detection of anti-inflammatory compounds

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used medications. But even if they help to relieve pain and swelling caused by inflammation, they can also cause side effects such as stomach irritations, nausea or allergic reactions. Therefore, there is a real interest in identifying alternative anti-inflammatory drugs, notably selective cyclooxygenase-2 (COX-2) inhibitors, called coxibs [1]. In order to accelerate the discovery of new drugs, researchers need fast, cost-effective, and reliable methods suitable for large screenings. In this context, we have developed a simple and sensitive assay for the rapid identification of COX-2 inhibitors. This test is based on the chromogenic reaction of a compound with the active enzyme on a thin-layer chromatographic plate, with or without prior chromatographic separation. After dot-blot or separation of the samples, the TLC plate is sprayed with a COX-2 solution and incubated for 10 minutes at 37°C. Then a mixture of arachidonic acid and the co-substrate N,N,N′,N′-tetramethyl-p- phenylenediamine (TMPD) is applied onto the plate. After a few minutes, TMPD is oxidized by the active enzyme into purple grey product; as a consequence, COX-2 inhibitors appear on the plate as clear spots against the coloured background [2]. Reagent concentrations have been optimized to assure a low-cost assay while maintaining good sensitivity (below microgram). This TLC assay enables the fast evaluation of individual compounds in complex mixtures, which is impossible with expensive 96-well kits. This newly developed technique will be useful to promote the discovery of anti-inflammatory drugs.

 

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Publication History

Article published online:
16 November 2023