A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity

BACKGROUND: Prostaglandin H(2 )synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H(2 )(PGH(2)) 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. METHODS: We 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. RESULTS: The 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). CONCLUSION: We 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.