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

Abstract

Background: 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. 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 microM, 1 mM) caused significant inhibition of spin-adduct formation (72 +/- 11 and 100 +/- 16% inhibition of control respectively; P < 0.05). Indomethacin (100 microM) 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.

Figure 1

Schematic diagram showing the peroxidase activity of PGHS. The process requires prior formation of a tyrosine radical from a tyrosine residue in close proximity to the haem group (Tyr 385). The tyrosyl radical is either recycled or participates in the suicide inactivation of the enzyme (for review of this process see [39]. Following incorporation of oxygen and formation of PGG₂, the peroxidase reduces the peroxyl moiety to the equivalent alcohol. The process allows for the concomitant oxidation of spin-trap CPH to CP which is detected by EPR.

Figure 2

(a) Sample EPR spectra obtained in the absence (control; PGHS + AA) and presence of aspirin (100 μM or 1 mM) after correction for background autoxidation. EPR settings: B0-field, 3356 gauss; sweep width; 50 Gauss, sweep time, 30 sec; modulation amplitude, 1500 mGauss; microwave power, 20 mW. (b) Mean data for development of EPR signal intensity (AU) with time in the absence (control; PGHS + AA) and presence of aspirin (100 μM). In both cases, substrate (AA) was added at t = 0 min. P = 0.02, 2-way ANOVA, repeated measures: n = 9–12.

Figure 3

Effect of aspirin and salicylic acid (10 μM – 1 mM) on EPR signals generated from PGHS-1 after treatment with substrate (AA). In each case, incubations with aspirin or SA were for 10 min prior to the baseline EPR reading (t = -2 min, not shown). AA was added at t = 0 min and readings shown were taken at t = 1.5 min. *P < 0.05, **P < 0.01; 1-way ANOVA with Dunnett's post-hoc test vs. control: n = 8–10.

Figure 4

Comparative effects of SA and recognized NSAIDs (all 100 μM) on EPR signal intensity measured at t = 1.5 min. *P < 0.05, **P < 0.01; 1-way ANOVA with Dunnett's post-hoc test vs. control: n = 6–10.