Non-steroidal Cyclooxygenase (COX) InhibitorsChandra Mohan, Ph.D. EMD Chemicals, San Diego, CA 92121

Cyclooxygenases (COXs), also known as prostaglandin H synthases, are fatty acid oxygenases that contain about 600 amino acid residues and act on arachidonic acid to generate prostaglandins (PG). All vertebrates contain two COX genes: one encoding the constitutive COX-1 and another inducible COX-2. COX-1 and COX-2 share approximately 60-65% amino acid identity. These COX isoforms are bifunctional hemoproteins that catalyze both the bioxygenation of arachidonic acid to form PGG2 and the peroxidative reduction of PGG2 to form PGH2. Hence, the catalytic domain of COX is considered to contain both cyclooxygenase and peroxidase active sites. The peroxidase site is required for the activation of heme groups that participate in the cyclooxygenase reaction. The active site is reported to be about 8 Å wide and 25 Å long that opens in the membrane-binding domain and accepts the arachidonic acid released from the membrane by phospholipases. The amino terminus of COX contains a single epidermal growth factor (EGF) module with conserved disulfide bonds, which functions as a dimerization domain. Following this domain are four amphipathic helices that anchor COX to the cell membrane. The active site of COX-2 is slightly larger and can accommodate bigger structures than those that are able to reach the active site of COX-1.

COX-1 is an ubiquitously and constitutively expressed enzyme that is associated with the endoplasmic reticulum (ER). It is responsible for maintaining normal physiologic functions and is considered as a “housekeeping” enzyme. It is normally expressed in the gastrointestinal tract, kidneys, and platelets and appears to be responsible for mediating the production of thromboxane A2 and prostaglandins. COX-2 is an inducible enzyme and is generally present at very low levels. It is mainly associated with the nuclear envelope and is primarily associated with inflammation. Cytokines and growth factors increase the expression of COX-2, mainly at inflammatory sites, producing prostaglandins,

which mediate inflammation, pain, and fever. The lumen of the ER is important for both the structure and function of COXs: its oxidative potential allows formation of the disulfide bonds of the enzymes and the N-linked glycosylation,

InflammatorySignaling

PLA2Activation

ArachidonicAcid

PGG2

Prostaglandins

Hypertension

Inflammation

PlateletAggregation

Thromboxane

PainFever

COX

COX

which occurs in the ER, appears to be necessary for proper folding of the enzyme. The newly synthesized COX is activated at Tyr384 (in human COX-1) or at Tyr371 (in human COX-2) to produce a tyrosyl radical, which converts arachidonic acid to an arachidonyl radical, which in-turn then reacts with two molecules of oxygen to yield PGG2. PGG2 diffuses to the peroxidase site where it is reduced to form PGH2. COXs have a short catalytic half-life because of the fact that the enzyme is auto-inactivated.

Most non-steroidal anti-inflammatory drugs (NSAIDs) exert anti-inflammatory and analgesic effects through the inhibition of prostaglandin synthesis by blocking COX activity. Traditional NSAIDs inhibit prostaglandin formation through the inhibition of both COX-1 and COX-2. Inhibition of COX-1 is not necessary for anti-inflammatory and analgesic effects, but is thought to account for much of the toxicity of traditional NSAIDs. Based on structural differences in the active sites of these two isozymes, several new drugs have been developed that specifically inhibit only the COX-2 activity. COX-2 inhibitors have the potential to provide the traditional benefits of NSAID with significantly reduced incidence of endoscopic ulcers. Hence, they offer greater therapeutic promise in the treatment of inflammation and cancer. The selective COX-2 inhibitors have great clinical significance because they can allow the preservation of COX-1 activity, which is essential in maintaining prostaglandins that are important for normal platelet function and protection of the gastrointestinal mucosa, and still inhibit COX-2 to reduce

inflammation and other pathologic processes. Increased expression of COX-2 has been associated with increased incidence of colon and breast cancers. It is over-expressed in about 50% of adenomas and 85% of adenocarcinomas of the colon. Hence, COX-2 inhibitors offer greater therapeutic promise not only in the treatment of inflammation, but also some forms of cancer. COX-2 inhibitors are also shown to augment photosensitization by increasing apoptosis and improve the responsiveness of photodynamic therapy.

More recently, there has been an upsurge of interest in COX-2 inhibitors as possible candidates for the treatment of Alzheimer’s disease. This is due to the fact that researchers have begun to think about “inflammation as a factor” in the development and/or progression of Alzheimer’s disease. Studies have shown that aggregated synthetic A1-40 peptide induces COX-2 expression in SH-SY5Y neuroblastoma cells and also stimulates oxygenase and peroxidase activities of COX-2 in a cell free system. Since neuronal excitation and oxidative stress have been linked to the pathogenesis of several neurodegenerative disorders, inhibiting excessive COX-2 activity may reduce the oxidative stress-induced neuronal damage and trauma. Several epidemiological studies have indeed shown that groups of people on NSAIDs, for unrelated conditions, such conditions as rheumatoid arthritis, have reduced incidence of Alzheimer’s disease. NSAIDs are believed to inhibit human A aggregation in vitro and reverse the -sheet conformation of preformed fibrils.

References:Nivsarkar, M., et al. 2008. Pharmacol. Reports 60, 692.

Ho, L., et al. 2006. CNS Drugs 20, 85.

Mazhar, D., et al. 2006. Br. J. Cancer 94, 346.

Ferrario, A., et al. 2005. Cancer Res. 65, 9473.

Hawkey, C.J., and Fortun, P.J. 2005. Curr. Opin. Gastroenterol. 21, 660.

Patrignani, P., et al. 2005. Brain Res. Brain Res. Rev. 48, 352.

Chandrashekharan, N.V., and Simmons, D.L. 2004. Genome Biol. 5, 241.

McGeer, P.L., and McGeer, E.G. 2001. Neurobiol. Aging 22, 799.

Thomas, T., et al. 2001. Neuroreport 12, 3263.

Smith, W.L., et al. 2000. Ann. Rev. Biochem. 69, 145.

Pasinetti, G.M., and Aisen, P.S. 1998. Neuroscience 87, 319.

Product Cat. No. Description Size

Anti-COX2, C-Terminal Rabbit pAb 236002Immunoaffinity purified. Immunogen used was a synthetic peptide corresponding to a 17-amino acid sequence near the C-terminus of human COX2. Reacts with bovine, human, mouse, rat, sheep. Does not cross-react with COX1. Suitable for ELISA and immunoblotting.

50 μg

Anti-COX2 Mouse mAb (5E10/D10) 236004Immunoaffinity purified. Immunogen used was purified COX2 from HL-60 cells. Recognizes COX2. in human and mouse. Does not cross-react with COX1. Suitable for ELISA and immunoblotting.

100 μg

Antibodies for Cyclooxygenases

Cyclooxygenase (COX) Inhibitors

Product Cat. No. Description SizeCOX-1 Inhibitor, FR122047(4,5-Bis(4-methoxyphenyl-2-[(1-methylpiperazin-4-yl)carbonyl]thiazole, HCl)

236005A cell-permeable, potent, and selective inhibitor of COX-1 (IC50 = 28 nM) with much lower selectivity for COX-2 (IC50 = 65 μM).

5 mg

COX-1 Inhibitor II(4-Amino-(N-(4-cholorophenyl)-N-methyl)benzenesulfonamide)

236006A cell-permeable, catalytic site targeting, COX-1-selective inhibitor (IC50 = 3.2 μM and >100 μM for COX-1 and COX-2, respectively).

25 mg

COX-1 Inhibitor IV, TFAPN-(5-Amino-2-pyridinyl)-4-trifluoromethylbenzamide

236007A cell-permeable compound that acts as a COX-1-selective inhibitor (IC50 = 0.80 and 210 M against ovine COX-1 and COX-2, respectively).

10 mg

COX-2 Inhibitor I(Methyl [5-methylsulfonyl-1-(4-chlorobenzyl)-1H-2-indolyl]carboxylate)

236011 A cell-permeable, selective inhibitor of COX-2 (IC50 = 650 nM) over COX-1 (IC50 > 10 M). 5 mg

COX-2 Inhibitor II (4-[(5-Difluoromethyl-3-phenyl)-4-isoxazolyl]benzenesulfonamide)

236012A cell-permeable, potent, and highly selective inhibitor of COX-2 both in vitro (IC50 = 4 nM for hCOX-2 vs. 114 μM for hCOX-1) and in vivo.

5 mg

COX-2 Inhibitor III(N-(5-Acetyl-2-piperidinophenyl)-N’-(2,5-dichlorophenyl)thiourea)

236013An active-site-targeting COX-2 inhibitor that exhibits better selectivity than Nimesulide and Celecoxib. (% inhibition of COX-1 and COX-2 at 10.0 μM inhibitor concentration = 5.1 and 26.36%, respectively).

25 mg

COX-2 Inhibitor IV(5-(4-Methoxyphenyl)-3,7-dimethyl-4,5-dihydroisoxazolo[4,5-d]pyridazin-4-one)

236014An active-site-targeting COX-2 inhibitor with potency and selectivity over COX-1 (33.85% inhibition of COX-2 at 10.0 μM).

25 mg

COX-2 Inhibitor V, FK3311(4’-Acetyl-2’-(2,4-Difluorophenoxy) methanesulfonanilide)

236015A cell-permeable, COX-2-selective inhibitor that with 500-fold greater potency in inhibiting the LPS-induced TxB2 production (IC50 = 316 nM) compared to the non-induced/constitutive TxB2 (IC50 = 160 μM).

5 mg

Curcumin, Curcuma longa L.[1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione]

239802A cell-permeable and irreversible antitumor and anti-inflammatory agent that acts as an inhibitor of 5-lipoxygenase (IC50 = 8 M) and cyclooxygenase (IC50 = 52 M).

100 mg

Diclofenac, 4’-Hydroxy-(2-[((2’,6’-Dichloro-4’-hydroxy) phenyl)amino]benzeneacetic Acid)

287845A cell-permeable metabolite of Diclofenac that act as a potent inhibitor of COX 2 (IC50 = 16.9 nM).

100 g

Diclofenac Sodium(2-[(2,6-Dichlorophenyl)amino]benzeneacetic Acid, Sodium)

287840A cell-permeable, non-selective COX inhibitor (IC50 = 60 nM and 200 nM for ovine COX-1 and COX-2 respectively).

1 g

DuP-697(5-Bromo-2-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)thiophene)

317500A cell permeable, potent, irreversible, and time-dependent COX-2 inhibitor with over 50-fold greater inhibitory potency against human and murine recombinant COX-2 (IC50 = 80 nM and 40 nM at 5 and 10 min respectively) than COX-1 (IC50 = 9 μM).

5 mg

Ebselen(2-Phenyl-1,2-benzisoselenazol-3(2H)-one)

324483A cell-permeable, seleno-organic compound that acts as a non-selective inhibitor of both COX-1 and COX-2.

5 mg

ETYA(5,8,11,14-Eicosatetraynoic Acid)

434741Cell permeable. Inhibits cyclooxygenase (IC50 = 8 M) and several lipooxygenases in whole cells. Also acts as a potent modulator of Ca2+ entry into cells.

20 mg

Flurbiprofen((±)-2-Fluoro-a-methyl[1,1’-biphenyl]-4-acetic Acid)

344079A mixture of S(+) and R(–) enantiomers. A cell-permeable, potent and non-selective inhibitor of cyclooxygenases.

100 mg

(±)-Ibuprofen 401003A reversible and competitive inhibitor of COX-1 (IC50 = 4.85 μM). Inhibits COX-2 at higher concentrations (IC50 = 223 μM).

1 g

Indomethacin(1-(p-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic Acid)

405268A cell permeable, non-selective inhibitor of COX (IC50 = 740 nM for COX-1; IC50 = 970 nM for COX-2).

10 g

Indomethacin Ester, 4-Methoxyphenyl-(1-(p-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic Acid, 4-Methoxyphenyl Ester)

405271A cell-permeable 4-methoxyphenyl ester derivative of Indomethacin that acts as a potent and selective inhibitor of COX-2 (IC50 = 40 nM) compared to COX-1 (IC50 > 66 μM).

5 mg

Kaempferol(3,4’5,7-Tetrahydroxyflavone)

420345A cell-permeable inhibitor of COX-1 activity (IC50 = 180 μM). Also blocks the transcriptional activation of COX-2 (IC50 <15 μM).

25 mg

MEG Hydrochloride(Mercaptoethylguanidine, HCl)

444600A cell-permeable inhibitor of inducible nitric oxide synthase (iNOS) and a peroxynitrite scavenger that also causes a dose-dependent inhibition of COX-1 (IC50 = 20 μM) and COX-2 activity.

10 mg

Meloxicam(4-Hydroxy-2-methyl-N-(5-methyl-2-thiazoyl)-2H-1, 2-benzothiazine-3-carboxamide-1,1-

444800A cell-permeable, selective inhibitor of COX-2 (IC50 = 4.7 μM) compared to COX-1 (IC50 = 36.6 μM).

100 mg

NPPB(5-Nitro-2-(3-phenylpropyl-amino)benzoic Acid)

484100Potent Cl- channel blocker (IC50 = 100 nM-100 M), depending on channel subtype and assay method. Inhibits cyclooxygenase (IC50 = 8 M).

10 mg

NS-398(N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulfonamide

349254A cell-permeable, selective inhibitor of COX-2 (IC50 = 3.8 μM). Does not affect COX-1 activity even at 100 μM.

5 mg

Product Cat. No. Description Size

COX-2 ELISA KitCBA053

A 96-well complete kit for the quantitative determination of human COX-2 in cell lysates. The 96-well plate is coated with a rabbit anti-human COX-2 polyclonal antibody as the capture antibody. Standards and samples are added to the wells at which time COX-2 is bound to the coated antibody. COX-2 is then detected with a monoclonal anti-COX-2 antibody and a goat anti-mouse IgG HRP Conjugate. The addition of TMB Substrate results in a blue-colored product in the presence of the HRP Conjugate. Sensitivity is increased by the addition of ELISA Stop Solution (H2SO4), yielding a yellow color. The absorbance of each well is measured at 450 nm (preferably with reference wavelength set of 540-600 nm).

1 kit

Also Available

Buy a Set and Save

Product Cat. No. Description Size

Cyclooxygenase Inhibitor Set 239783

The set contains: 100 mg of Meloxicam (Cat. No. 444800)5 mg of NS-398 (Cat. No. 349254) 5 mg of SC-560 (Cat. No. 565610) 5 mg of Sulindac Sulfide (Cat. No. 574102)

1 set

Cyclooxygenase (COX) Inhibitors Continued

Product Cat. No. Description Size

Parthenolide, Tanacetum parthenium 512732A cell-permeable inhibitor of COX-2 and proinflammatory cytokines (TNF- and IL-1) expression in LPS-stimulated macrophages.

50 mg

Pterostilbene, Pterocarpus marsupium(trans-3,5-Dimethoxy-4’-hydroxystilbene)

523310A cell-permeable methoxylated analog of Resveratrol (Cat. No. 554325) that moderately inhibits COX-1 and -2 activities (IC50 = 19.8 μM and 83.9 μM) respectively.

10 mg

Radicicol, Diheterospora chlamydosporia 553400An antifungal macrocyclic lactone antibiotic that inhibits the expression of COX-2 (IC50 = 27 nM) without affecting COX-1 expression in LPS-stimulated macrophages.

500 g

Resveratrol(trans-3,4’,5-Trihydroxystilbene)

554325A specific inhibitor of COX-1 (ED50 = 15 μM). Also inhibits the hydroxyperoxidase activity of COX-1 (ED50 = 3.7 μM).

25 mg

SC-560(5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole)

565610A cell-permeable, highly potent, and selective inhibitor of COX-1 (IC50 = 9 nM) over COX-2 (IC50 = 6.3 M).

5 mg

Sodium Salicylate 567630 A cell-permeable weak inhibitor of COX-2 (IC50 = 100 M). 5 g

Sulindac Sulfide((Z)-5-Fluoro-2-methyl-1-[p-(methylthio)benzylidene]indene-3-acetic Acid)

574102 A cell-permeable, selective, potent inhibitor of COX-1 (ID50 = 500 nM) over COX-2 (IC50 = 14 nM). 5 mg

Sulindac Sulfone((Z)-5-Fluoro-2-methyl-1[p-(methylsulfonyl) benzylidene]indene-3-acetic Acid)

574105A cell-permeable sulfone metabolite of Sulindac (Cat. No. 574100) that has anticancer properties but lacks COX inhibitory activity.

5 mg

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2.5

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0 50 100 150 200 250Cox-2 (ng/ml)

Abs

orba

nce

450

nm COX-2 ELISA Kit Standard Curve

A standard curve was generated according to the COX-2 Standard preparation and the detailed protocol included in the kit. The results represent a typical standard curve. Assay range: 2.5-200 ng/ml. Lower limit of detection: ~2 ng/ml.

Assay Range: 2.5-200 ng/ml

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For more information, visit www.emdbiosciences.com/CoxBulletin