Inhibitors Of PGHS-1 Research Articles

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

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.

Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H 2 synthases by ibuprofen, naproxen, and indomethacin

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [ S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) α and γ isoforms in CV-1 cells co-transfected with rat PPAR α and γ, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA β-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid β-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARα activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(−)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARγ, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(−)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(−)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(−)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARα and/or PPARγ isoforms.

Paradoxical effects of resveratrol on the two prostaglandin H synthases

Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS, PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol, a lignan, was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K I of 26 ± 2 μM but enhanced the PGHS-2 activity nearly twofold. Additionally, resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC 50 = 15 μM) better than that of PGHS-2 (IC 50 = >200 μM). Inhibition of the peroxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG 2 from arachidonic acid. A plausible relationship between these observations and the anticarcinogenic activity of resveratrol is discussed.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.