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• W2783503367 abstract "The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H2 (PGH2). COX-2 also oxygenates the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA) to the corresponding PGH2 analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine. Here, we investigated the mechanism of COX inhibition by ARN2508, an NSAID that inhibits both COXs and FAAH with high potency, target selectivity, and decreased gastrointestinal toxicity in mouse models, presumably due to its ability to increase levels of FAEs. A 2.27-Å–resolution X-ray crystal structure of the COX-2·(S)-ARN2508 complex reveals that ARN2508 adopts a binding pose similar to that of its parent NSAID flurbiprofen. However, ARN2508's alkyl tail is inserted deep into the top channel, an active site region not exploited by any previously reported NSAID. As for flurbiprofen, ARN2508's potency is highly dependent on the configuration of the α-methyl group. Thus, (S)-ARN2508 is more potent than (R)-ARN2508 for inhibition of AA oxygenation by both COXs and 2-AG oxygenation by COX-2. Also, similarly to (R)-flurbiprofen, (R)-ARN2508 exhibits substrate selectivity for inhibition of 2-AG oxygenation. Site-directed mutagenesis confirms the importance of insertion of the alkyl tail into the top channel for (S)-ARN2508's potency and suggests a role for Ser-530 as a determinant of the inhibitor's slow rate of inhibition compared with that of (S)-flurbiprofen. The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H2 (PGH2). COX-2 also oxygenates the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA) to the corresponding PGH2 analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine. Here, we investigated the mechanism of COX inhibition by ARN2508, an NSAID that inhibits both COXs and FAAH with high potency, target selectivity, and decreased gastrointestinal toxicity in mouse models, presumably due to its ability to increase levels of FAEs. A 2.27-Å–resolution X-ray crystal structure of the COX-2·(S)-ARN2508 complex reveals that ARN2508 adopts a binding pose similar to that of its parent NSAID flurbiprofen. However, ARN2508's alkyl tail is inserted deep into the top channel, an active site region not exploited by any previously reported NSAID. As for flurbiprofen, ARN2508's potency is highly dependent on the configuration of the α-methyl group. Thus, (S)-ARN2508 is more potent than (R)-ARN2508 for inhibition of AA oxygenation by both COXs and 2-AG oxygenation by COX-2. Also, similarly to (R)-flurbiprofen, (R)-ARN2508 exhibits substrate selectivity for inhibition of 2-AG oxygenation. Site-directed mutagenesis confirms the importance of insertion of the alkyl tail into the top channel for (S)-ARN2508's potency and suggests a role for Ser-530 as a determinant of the inhibitor's slow rate of inhibition compared with that of (S)-flurbiprofen. The enzyme cyclooxygenase (COX) 3The abbreviations used are: COXcyclooxygenaseAAarachidonic acidPGprostaglandin2-AG2-arachidonoylglycerolAEAarachidonoylethanolamideFAEfatty acyl ethanolamideFAAHfatty acid amide hydrolaseNSAIDnon-steroidal anti-inflammatory drugmCOX-2murine cyclooxygenase-2oCOX-1ovine cyclooxygenase-1LNAα-linolenic acidPGE2-GPGE2-glycerol esterEPPS4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid. catalyzes the committed step in prostanoid biosynthesis, the bis-dioxygenation and cyclization of AA to form the endoperoxy-hydroperoxide intermediate prostaglandin (PG) G2. Then, via its peroxidase activity, the enzyme reduces the 15-hydroperoxy group of PGG2 to produce the final product, PGH2 (1Rouzer C.A. Marnett L.J. Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases.Chem. Rev. 2003; 103 (12797830): 2239-2304https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). PGH2 is a substrate for various terminal prostanoid synthases that catalyze the formation of PGs as well as thromboxane A2. PGs exert various biological effects including platelet aggregation, gastrointestinal and cardiovascular regulation, parturition, and modulation of the inflammatory response (2Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. Cyclooxygenase in biology and disease.FASEB J. 1998; 12 (9737710): 1063-1073Crossref PubMed Scopus (2221) Google Scholar). cyclooxygenase arachidonic acid prostaglandin 2-arachidonoylglycerol arachidonoylethanolamide fatty acyl ethanolamide fatty acid amide hydrolase non-steroidal anti-inflammatory drug murine cyclooxygenase-2 ovine cyclooxygenase-1 α-linolenic acid PGE2-glycerol ester 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid. There are two COX isoforms, COX-1 and COX-2, distinguished primarily by their patterns of expression. Specifically, the expression of COX-2 is induced in response to proinflammatory cytokines and growth factors, whereas the expression of COX-1 is constitutive in most tissues (3Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69 (10966456): 145-182https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In addition, because of its larger active site, COX-2 can oxygenate analogs of AA that are poor substrates for COX-1. These include the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA), oxygenation of which yields PGH2-glyceryl ester and PGH2-ethanolamide, respectively (4Kozak K.R. Rowlinson S.W. Marnett L.J. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2.J. Biol. Chem. 2000; 275 (10931854): 33744-33749https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 5Yu M. Ives D. Ramesha C.S. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2.J. Biol. Chem. 1997; 272 (9261124): 21181-21186https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 6Kozak K.R. Crews B.C. Morrow J.D. Wang L.H. Ma Y.H. Weinander R. Jakobsson P.J. Marnett L.J. Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides.J. Biol. Chem. 2002; 277 (12244105): 44877-44885https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Both COX enzymes, but particularly COX-2, are the major sites of action of the widely used non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, indomethacin, and naproxen (7Blobaum A.L. Marnett L.J. Structural and functional basis of cyclooxygenase inhibition.J. Med. Chem. 2007; 50 (17341061): 1425-1441https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). A major limitation to the use of most NSAIDs is gastrointestinal toxicity due to blockade of the biosynthesis of cytoprotective PGs (8Scarpignato C. Hunt R.H. Nonsteroidal antiinflammatory drug-related injury to the gastrointestinal tract: clinical picture, pathogenesis, and prevention.Gastroenterol. Clin. North Am. 2010; 39 (20951911): 433-464https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 9Sostres C. Gargallo C.J. Lanas A. Nonsteroidal anti-inflammatory drugs and upper and lower gastrointestinal mucosal damage.Arthritis Res. Ther. 2013; 15 (24267289): S3https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). This problem was addressed by the development of COX-2–selective inhibitors that preserved COX-1–dependent gastrointestinal PGE2 production. These drugs exhibit good anti-inflammatory activity with reduced gastrointestinal side effects; however, subsequent clinical trials uncovered adverse cardiovascular side effects associated with inhibition of COX-2 (10Capone M.L. Tacconelli S. Rodriguez L.G. Patrignani P. NSAIDs and cardiovascular disease: transducing human pharmacology results into clinical read-outs in the general population.Pharmacol. Rep. 2010; 62 (20631418): 530-535https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar, 11Marnett L.J. The COXIB experience: a look in the rearview mirror.Annu. Rev. Pharmacol. Toxicol. 2009; 49 (18851701): 265-290https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). An alternative approach to NSAID-mediated gastrointestinal toxicity is to increase the levels of AEA and other fatty acyl ethanolamides (FAEs), such as palmitoylethanolamide, in individuals receiving NSAID therapy. AEA and palmitoylethanolamide exert anti-inflammatory and cytoprotective effects in the gastrointestinal tract that can compensate for loss of beneficial PGs (12Sanger G.J. Endocannabinoids and the gastrointestinal tract: what are the key questions?.Br. J. Pharmacol. 2007; 152 (17767170): 663-670https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar). However, during inflammation, this FAE-dependent signaling may be compromised through increased expression of fatty acid amide hydrolase (FAAH), the primary degradative enzyme for FAEs, and COX-2, which may also contribute to AEA depletion because of its oxidation. These considerations have led to the hypothesis that simultaneous blockade of COX and FAAH might promote FAE-mediated prevention of the gastrointestinal side effects of NSAIDs while preserving most of their anti-inflammatory effects. To test this hypothesis, dual inhibitors that simultaneously target both the COX enzymes and FAAH with high potency and oral bioavailability were synthesized. One compound from this class, ARN2508 (see Fig. 1), decreases intestinal inflammation and protects the gastrointestinal tract from NSAID-induced toxicity in vivo (13Sasso O. Migliore M. Habrant D. Armirotti A. Albani C. Summa M. Moreno-Sanz G. Scarpelli R. Piomelli D. Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.FASEB J. 2015; 29 (25757568): 2616-2627https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). ARN2508 combines key structural features of the compound URB597, an FAAH inhibitor, and flurbiprofen (Fig. 1), a member of the 2-arylpropionic acid class of NSAIDs. Like flurbiprofen, ARN2508 inhibits PGE2 formation in the gastric mucosa, but unlike flurbiprofen, ARN2508 was found to protect the epithelial lining in the stomach of mice, likely through its ability to increase levels of AEA and other FAEs. Inhibition of both FAAH and COX by ARN2508 was found to be functionally irreversible based on dialysis experiments (13Sasso O. Migliore M. Habrant D. Armirotti A. Albani C. Summa M. Moreno-Sanz G. Scarpelli R. Piomelli D. Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.FASEB J. 2015; 29 (25757568): 2616-2627https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In the case of FAAH, ARN2508-mediated inhibition was attributed to a covalent bond formed between the carbamate moiety of the inhibitor and the enzyme's catalytic serine (14Mileni M. Kamtekar S. Wood D.C. Benson T.E. Cravatt B.F. Stevens R.C. Crystal structure of fatty acid amide hydrolase bound to the carbamate inhibitor URB597: discovery of a deacylating water molecule and insight into enzyme inactivation.J. Mol. Biol. 2010; 400 (20493882): 743-754https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Thus, the carbamate functional group of ARN2508 is required for FAAH inhibition; however, it is not necessary for inhibition of COX. In fact, the carboxylate moiety is necessary for COX inhibition by ARN2508 as is seen in the case of its parent compound, flurbiprofen, which inhibits COX by a noncovalent mechanism. These findings suggest that covalent modification is not required for ARN2508-mediated inhibition of COX, but they do not completely rule out this possibility. Flurbiprofen contains one chiral center α to the carboxylic acid. Potent COX inhibitory activity is associated only with the S-enantiomer of flurbiprofen; however, the R-enantiomer is a substrate-selective inhibitor of endocannabinoid oxygenation by COX-2 (15Duggan K.C. Hermanson D.J. Musee J. Prusakiewicz J.J. Scheib J.L. Carter B.D. Banerjee S. Oates J.A. Marnett L.J. (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2.Nat. Chem. Biol. 2011; 7 (22053353): 803-809https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). ARN2508 shares this chiral carbon, but the initial kinetic evaluation of its COX-inhibitory activity was carried out using the racemic mixture (13Sasso O. Migliore M. Habrant D. Armirotti A. Albani C. Summa M. Moreno-Sanz G. Scarpelli R. Piomelli D. Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.FASEB J. 2015; 29 (25757568): 2616-2627https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Here, we report the characterization of the S- and R-enantiomers of ARN2508 with regard to potency, time dependence, isoform selectivity, and substrate selectivity of COX inhibition. We also explore structural determinants of ARN2508-mediated inhibition of COX and report the crystal structure of COX-2 complexed with (S)-ARN2508. Our findings reveal that the enantioselectivity of ARN2508 is similar to that of flurbiprofen. Also, like (S)-flurbiprofen, (S)-ARN2508 is a time-dependent inhibitor of COX-2, although its rate of inhibition is substantially slower than that of its parent. Furthermore, whereas the flurbiprofen moiety of ARN2508 binds in the COX-2 active site in a pose very similar to that of flurbiprofen, the inhibitor's alkyl chain occupies the same channel of COX-2 as the ω-tail of AA in a region of the active site that has not been exploited by any other NSAID for which structural data are available. Thus, in addition to its novel pharmacology as a dual inhibitor of FAAH and COX, ARN2508 exhibits binding properties not previously observed for any of the numerous selective and nonselective COX inhibitors described previously. To better understand the basis for inhibitor binding, we obtained the three-dimensional structure of COX-2 in complex with (S)-ARN2508 via X-ray crystallography by molecular replacement using the high-resolution monomer model of mCOX-2 in complex with naproxen (Protein Data Bank code 3NT1) (16Duggan K.C. Walters M.J. Musee J. Harp J.M. Kiefer J.R. Oates J.A. Marnett L.J. Molecular basis for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen.J. Biol. Chem. 2010; 285 (20810665): 34950-34959https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The COX-2·(S)-ARN2508 complex was determined to 2.27-Å resolution in the space group of I4122. The omit difference map (Fo − Fc) clearly revealed that the ligand in the crystal is indeed the S-enantiomer of ARN2508 (Fig. 2 and Table 1). We have not been able to obtain high quality crystals of the COX-2·(R)-ARN2508 complex, possibly due to the low potency of this inhibitor.Table 1X-ray data collection and refinement statisticsCOX-2·(S)-ARN2508 (Protein Data Bank code 5W58)Data collectionWavelength (Å)0.9792Resolution range (Å)66.05–2.27 (2.35–2.27)Space groupI4122Unit cell (a, b, c)173.8, 173.8, 203.3Total reflections1,048,952 (101,264)Unique reflections71,733 (6,799)Multiplicity14.6 (14.4)Completeness (%)0.99 (0.99)Mean I/σ(I)19.41 (0.82)Wilson B-factor (Å2)48.02Rmerge0.1551 (2.12)CC1/20.999 (0.336)RefinementRwork/Rfree16.9/18.9 (33.3/33.4)Number of atoms (total/protein/ligands/solvents)4,967/4,512/181/275r.m.s. (bond/angle)0.012/1.26Ramachandran favored/outliers (%)98.0/0Average B-factor (total/protein/ligands/solvents)55.86/55.06/75.81/55.92 Open table in a new tab The cyclooxygenase active site of COX-2 comprises a long L-shaped channel beginning at the membrane-binding domain and terminating deep within the catalytic domain. The active site is separated from the membrane-binding domain and an enlarged “lobby” immediately above it by a constriction formed by Arg-120, Tyr-355, and Glu-524. Above the constriction, the channel is lined predominantly by hydrophobic residues that favor binding of fatty acid substrates to the active site. Notable exceptions are Tyr-385, the catalytic residue that, in radical form, initiates the first step in PG biosynthesis, and Ser-530. Both of these residues are located at the bend in the channel (Fig. 3). AA binds in this channel with the carboxyl group in close proximity to Arg-120 and Tyr-355 at the constriction site, carbon 13 aligned with the catalytic Tyr-385, and the ω-tail deep in the alcove above Ser-530 (17Malkowski M.G. Ginell S.L. Smith W.L. Garavito R.M. The productive conformation of arachidonic acid bound to prostaglandin synthase.Science. 2000; 289 (10988074): 1933-1937https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 18Vecchio A.J. Simmons D.M. Malkowski M.G. Structural basis of fatty acid substrate binding to cyclooxygenase-2.J. Biol. Chem. 2010; 285 (20463020): 22152-22163https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Flurbiprofen also binds in the active site with the carboxylate close to the polar constriction site residues; however, this more compact molecule does not extend into the active site above Ser-530 (19Gupta K. Selinsky B.S. Loll P.J. 2.0 Å structure of prostaglandin H2 synthase-1 reconstituted with a manganese porphyrin cofactor.Acta Crystallogr. D Biol. Crystallogr. 2006; 62 (16421446): 151-156https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 20Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents.Nature. 1996; 384 (8967954): 644-648https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 21Picot D. Loll P.J. Garavito R.M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1.Nature. 1994; 367 (8121489): 243-249https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 22Selinsky B.S. Gupta K. Sharkey C.T. Loll P.J. Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations.Biochemistry. 2001; 40 (11318639): 5172-5180https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar, 23Sidhu R.S. Lee J.Y. Yuan C. Smith W.L. Comparison of cyclooxygenase-1 crystal structures: cross-talk between monomers comprising cyclooxygenase-1 homodimers.Biochemistry. 2010; 49 (20669977): 7069-7079https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). ARN2508 is an alkyl carbamic acid biphenyl conjugate. As might be predicted, the flurbiprofen moiety of ARN2508 sits in the hydrophobic channel of the enzyme's active site with the same binding mode that is adopted by flurbiprofen in COX-1 and COX-2 (19Gupta K. Selinsky B.S. Loll P.J. 2.0 Å structure of prostaglandin H2 synthase-1 reconstituted with a manganese porphyrin cofactor.Acta Crystallogr. D Biol. Crystallogr. 2006; 62 (16421446): 151-156https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 20Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents.Nature. 1996; 384 (8967954): 644-648https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 21Picot D. Loll P.J. Garavito R.M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1.Nature. 1994; 367 (8121489): 243-249https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 22Selinsky B.S. Gupta K. Sharkey C.T. Loll P.J. Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations.Biochemistry. 2001; 40 (11318639): 5172-5180https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar, 23Sidhu R.S. Lee J.Y. Yuan C. Smith W.L. Comparison of cyclooxygenase-1 crystal structures: cross-talk between monomers comprising cyclooxygenase-1 homodimers.Biochemistry. 2010; 49 (20669977): 7069-7079https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Thus, the carboxylate moiety of ARN2508 lies at the constriction of the active site, ion-pairing to Arg-120 and hydrogen-bonding to Tyr-355, and the aromatic rings project upward toward Tyr-385. However, hydrogen bond formation with ARN2508's carbamate group requires an upward displacement of the side chain of Ser-530 in the COX-2·(S)-ARN2508 complex relative to its position in the COX-2·flurbiprofen complex. Additional hydrogen bonds form between Tyr-385 and the oxygen atoms of ARN2508's carbamate group. The alkyl chain of ARN2508 reaches deeply into the distal portion of the cyclooxygenase channel between helices 2 and 17, making hydrophobic interactions with various residues including Phe-205, Phe-209, Phe-210, Val-228, Ile-341, Val-344, Ile-377, Phe-381, and Leu-534 (Fig. 2). It was previously reported that racemic ARN2508 has an IC50 value of ∼12 nm for COX-1 and 430 nm for COX-2 (13Sasso O. Migliore M. Habrant D. Armirotti A. Albani C. Summa M. Moreno-Sanz G. Scarpelli R. Piomelli D. Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.FASEB J. 2015; 29 (25757568): 2616-2627https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). To further assess the potency of the inhibitor, each enantiomer was individually tested for both COX-1 and COX-2 inhibition. The results (Fig. 4) indicated that the potency of the S-enantiomer of ARN2508 (IC50 = 7.0 nm) for COX-1 inhibition is much greater than that of the R-enantiomer (IC50 = 4.6 μm) (Table 2). Similarly, using 5 μm AA as substrate, the S-enantiomer was the more potent of the two against COX-2, achieving complete inhibition of AA oxygenation with an IC50 of ∼39 nm (Fig. 5A and Table 2), whereas the R-enantiomer inhibited the enzyme by ∼50% at 10 μm and failed to reach complete inhibition at the highest concentration tested (100 μm). Thus, as previously reported for the racemic mixture (13Sasso O. Migliore M. Habrant D. Armirotti A. Albani C. Summa M. Moreno-Sanz G. Scarpelli R. Piomelli D. Multitarget fatty acid amide hydrolase/cyclooxygenase blockade suppresses intestinal inflammation and protects against nonsteroidal anti-inflammatory drug-dependent gastrointestinal damage.FASEB J. 2015; 29 (25757568): 2616-2627https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), each enantiomer of ARN2508 exhibits a small degree of COX-1 selectivity. The potency of the S-enantiomer for 2-AG oxygenation by COX-2 (IC50 = 21 nm) was greater than that for AA oxygenation but only by ∼2-fold (Fig. 5B and Table 2). In contrast, (R)-ARN2508 exhibited an ∼30-fold higher inhibitor potency for 2-AG (IC50 = 0.34 μm) versus AA oxygenation and completely blocked 2-AG oxygenation. These results indicate that the R-enantiomer is a substrate-selective COX-2 inhibitor.Table 2(±)-ARN2508 inhibition IC50 valuesEnzymeSubstratePreincubationIC50(S)-ARN2508(R)-ARN2508minμmCOX-1 WTAA100.0074.6COX-2 WTAA100.03910COX-2 WTAA0n/aan/a, unable to obtain fitting due to incomplete enzyme inhibition.11COX-2 WT2-AG100.0210.34COX-2 WT2-AG0n/a0.54COX-2 Y355FAA100.09929COX-2 S530AAA100.026n/aCOX-2 S530TAA100.291n/aa n/a, unable to obtain fitting due to incomplete enzyme inhibition. Open table in a new tab Figure 5Inhibition of mCOX-2 with ARN2508 with or without preincubation. Oxygenation of 5 μm AA (A) or 5 μm 2-AG (B) by mCOX-2 was assessed by quantification of enzymatic product formation utilizing LC-MS/MS as described under “Experimental procedures.” Each enantiomer of ARN2508 was preincubated for 10 min (10 m) or added simultaneously (0 m) with substrate. Each substrate was allowed to react for 10 s before quenching with organic solvent containing deuterated internal standards. Results are the mean ± S.D. of triplicate determinations. Some error bars are shorter than the height of the symbol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As most highly potent COX inhibitors are time-dependent, initial experiments included an arbitrarily chosen 10-min preincubation period prior to substrate addition. To further explore the time dependence of ARN2508, various concentrations of each enantiomer were added simultaneously with either AA or 2-AG to COX-2, reactions were quenched after 10 s, and products were analyzed using LC-MS/MS. The data indicate that COX-2 inhibition by the R-enantiomer is essentially time-independent for both AA and 2-AG oxygenation as preincubation had minimal effect on the observed IC50 values or extent of inhibition observed. In contrast, COX-2 inhibition by the S-enantiomer is strongly time-dependent as indicated by IC50 values in the nm range for both AA and 2-AG oxygenation following preincubation but failure to reach even 50% inhibition in the absence of preincubation at the concentrations tested (Fig. 5, A and B, and Table 2). The vast majority of COX-2 inhibitors bind to the enzyme in a non-covalent manner, a notable exception being aspirin, which acetylates Ser-530 (24Roth G.J. Stanford N. Majerus P.W. Acetylation of prostaglandin synthase by aspirin.Proc. Natl. Acad. Sci. U.S.A. 1975; 72 (810797): 3073-3076https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar). Most time-dependent, non-covalent inhibitors exhibit kinetics consistent with a two-step binding model that includes rapid, reversible formation of an initial complex followed by a slower step that leads to a much stronger enzyme-inhibitor interaction (25Rome L.H. Lands W.E. Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs.Proc. Natl. Acad. Sci. U.S.A. 1975; 72 (1061075): 4863-4865https://doi.org/10.1074/jbc.M117.802058Abstract Full Text Full Text PDF PubMed Google Scholar). To assess the kinetic mechanism of ARN2508-mediated COX-2 inhibition, various concentrations of the S-enantiomer were preincubated with wildtype COX-2 for different time periods, and the activity of the enzyme was measured. A plot of enzyme activity versus time for each inhibitor concentration exhibited pseudo-first order kinetics (Fig. 6A), and the observed first order rate constants for each curve (kobs) were plotted against inhibitor concentration. The resulting data (Fig. 6B) failed to yield the hyperbolic curve that is expected from the two-step model. A possible explanation is that the two-step model is correct, but insufficient concentrations of inhibitor were used to fully delineate the hyperbola. Indeed, fitting of the data to this model yields a first step dissociation constant of 220 μm and inhibition rate constant of 41 min−1, corresponding to an overall kinetic efficiency (kon/KI) of 0.18 min−1·μm−1. These values are, at best, only estimates as evaluation of much higher inhibitor concentrations is necessary to fully define the curve. Unfortunately, solubility limitations prevented this evaluation. An alternative explanation is that the plot is linear rather than hyperbolic, suggesting that ARN2508 does not follow the typical two-step kinetic mechanism. In this case, the binding process would more likely occur in a single step, or if multistep, the first step is rate-limiting. The slope of the line (0.11 min−1·μm−1) provides an estimate of the second order rate constant for the binding of inhibit" @default.
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• W2783503367 title "Dual cyclooxygenase–fatty acid amide hydrolase inhibitor exploits novel binding interactions in the cyclooxygenase active site" @default.
• W2783503367 cites W1025607298 @default.
• W2783503367 cites W105152574 @default.
• W2783503367 cites W1520750411 @default.
• W2783503367 cites W1883558819 @default.
• W2783503367 cites W1969365385 @default.
• W2783503367 cites W1980990431 @default.
• W2783503367 cites W1983759759 @default.
• W2783503367 cites W1997761684 @default.
• W2783503367 cites W2002840016 @default.
• W2783503367 cites W2007504470 @default.
• W2783503367 cites W2010311152 @default.
• W2783503367 cites W2010967782 @default.
• W2783503367 cites W2012344270 @default.
• W2783503367 cites W2015101897 @default.
• W2783503367 cites W2019144812 @default.
• W2783503367 cites W2019177139 @default.
• W2783503367 cites W2023167233 @default.
• W2783503367 cites W2024175 @default.
• W2783503367 cites W2024715019 @default.
• W2783503367 cites W2028742202 @default.
• W2783503367 cites W2030850720 @default.
• W2783503367 cites W2049772907 @default.
• W2783503367 cites W2061592031 @default.
• W2783503367 cites W2063837435 @default.
• W2783503367 cites W2074514503 @default.
• W2783503367 cites W2086458197 @default.
• W2783503367 cites W2086514081 @default.
• W2783503367 cites W2097111776 @default.
• W2783503367 cites W2100840632 @default.
• W2783503367 cites W2102081214 @default.
• W2783503367 cites W2106430772 @default.
• W2783503367 cites W2109312812 @default.
• W2783503367 cites W2111134372 @default.
• W2783503367 cites W2114374331 @default.
• W2783503367 cites W2115350007 @default.
• W2783503367 cites W2124026197 @default.
• W2783503367 cites W2126853519 @default.
• W2783503367 cites W2127130852 @default.
• W2783503367 cites W2134201060 @default.
• W2783503367 cites W2135853706 @default.
• W2783503367 cites W2147017754 @default.
• W2783503367 cites W2149083834 @default.
• W2783503367 cites W2149233326 @default.
• W2783503367 cites W2168852688 @default.
• W2783503367 cites W2178742227 @default.
• W2783503367 cites W2180229411 @default.
• W2783503367 cites W2217045346 @default.
• W2783503367 cites W2275489002 @default.
• W2783503367 cites W2952210817 @default.
• W2783503367 cites W4248872320 @default.
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