Abstract
BackgroundProstaglandin H2 synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2) prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2) are the target for nonsteroidal anti-inflammatory drugs (NSAIDs).Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel in vitro electron paramagnetic resonance (EPR)-based assay for measuring the activity of PGHS-1.MethodsWe validated a novel in vitro PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH) to 3-carboxy-proxy (CP) under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR) spectrum.ResultsThe assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 μM, 1 mM) caused significant inhibition of spin-adduct formation (72 ± 11 and 100 ± 16% inhibition of control respectively; P < 0.05). Indomethacin (100 μM) also abolished the signal (114 ± 10% inhibition of control; P < 0.01). SA and the PGHS-2-selective inhibitor, NS398, failed to significantly inhibit spin-adduct generation (P > 0.05).ConclusionWe have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays.
Highlights
Prostaglandin H2 synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2) prior to formation of prostanoids that are important in inflammation
The electron paramagnetic resonance (EPR)-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays
It was determined that t = 1.5 min was an appropriate point at which to compare free radical generation between control and nonsteroidal anti-inflammatory drugs (NSAID)-treated PGHS-1, given that spin-adduct generation in response to arachidonic acid (AA) had peaked – subsequent adduct formation was at an equivalent rate in control and AA-treated samples and was likely to be due to non-specific auto-oxidation of CPH
Summary
Prostaglandin H2 synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2) prior to formation of prostanoids that are important in inflammation. Prostaglandins are derived from arachidonic acid (AA) in a pathway dependent on the PGHS (EC 1.14.99.1) family of enzymes, which are commonly known as cyclooxygenase (COX), referring to the first step of enzymatic activity. The enzyme contains two active sites: a COX site, where AA is converted into the hydroperoxy endoperoxide, prostaglandin G2 (PGG2), and a haem with peroxidase activity that reduces PGG2 to PGH2 (For review see [1]). The reduction of PGG2 by the peroxidase element generates the corresponding alcohol This reaction has previously been demonstrated to concurrently oxidise aminopyrine molecules to aminopyrine free radicals [2]. A spin-trapping agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH) is oxidised to 3-carboxy-proxy (CP), probably under the action of the peroxidase, in a similar fashion to that previously seen with aminopyrine (Fig. 1)
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