Bradley University, Chemistry and Biochemistry, Peoria, IL 61625
Recently we have been using a fluorescent-based Amplex Red assay to measure cyclooxygenase (COX) activity in the presence of an inhibitor, such as ibuprofen. This assay involves monitoring the concomitant conversion of non-fluorescent substrate Amplex Red to the fluorescent resorufin product that accompanies the conversion of arachidonic acid to prostaglandin H2. The latter reaction is catalyzed by the peroxidase domain of the cyclooxygenase enzymes. After reproducing consistent results with ibuprofen, we plan to screen substituted ∂-lactones synthesized by Brad Andersh’s research group for selective inhibition of COX-2 activity. Substituted ∂-lactones have received attention as potential selective COX-2 inhibitors since de Aguiar Amaral et al. determined that ∂-valerolactones aid in reducing inflammation and pain, and suggested they did so by inhibiting a crucial step in the prostaglandin biosynthetic pathway. The pharmaceutical interest in selective COX-2 inhibitors stems from the following reasons: 1) normal COX-2 activity has been shown to contribute to inflammation and pain and has been linked to arthritis, cancer, Alzheimer’s disease and other pathogenic diseases, and 2) inhibition of the COX-1 isoform contributes to undesirable gastrointestinal side effects.
We have demonstrated that the fluorescence signal due to resorufin production increases as COX-2 levels increase. Furthermore, we have shown that increasing ibuprofen levels result in a concomitant decrease in the fluorescence signal indicating inhibition of COX-2. In this regard, 59.2% inhibition was observed when 160 U of COX-2 enzyme were incubated in the presence of 50 mM ibuprofen. This fluorescent-based in vitro assay should allow us to quickly screen the substituted ∂-lactones generated in Andersh’s lab for COX-2 inhibitory potential.
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