{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T02:41:48Z","timestamp":1791513708268,"version":"4.3.4"},"reference-count":33,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5367","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,5,22]]},"abstract":"<jats:p>\n            Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was\n            <jats:italic>o<\/jats:italic>\n            -(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.\n          <\/jats:p>","DOI":"10.1126\/science.280.5367.1268","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:35:26Z","timestamp":1027762526000},"page":"1268-1270","source":"Crossref","is-referenced-by-count":227,"title":["Aspirin-like Molecules that Covalently Inactivate Cyclooxygenase-2"],"prefix":"10.1126","volume":"280","author":[{"given":"Amit S.","family":"Kalgutkar","sequence":"first","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brenda C.","family":"Crews","sequence":"additional","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott W.","family":"Rowlinson","sequence":"additional","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Garner","sequence":"additional","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karen","family":"Seibert","sequence":"additional","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lawrence J.","family":"Marnett","sequence":"additional","affiliation":[{"name":"A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, C. Garner, L. J. Marnett, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Cancer Center, Center in Molecular Toxicology, Departments of Biochemistry and Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA."},{"name":"K. Seibert, Searle\/Monsanto, 800 North Lindbergh (T3G), St. Louis, MO 63167, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","unstructured":"J. R. Vane R. J. Flower R. M. Botting Stroke 21 (suppl. 4) 12 (1990)."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1007\/BF01662127","volume":"76","author":"Dreser H.","year":"1899","unstructured":"Dreser H., Pfluegers Arch.76, 306 (1899).","journal-title":"Pfluegers Arch."},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"3073","DOI":"10.1073\/pnas.72.8.3073","volume":"72","author":"Roth G. J.","year":"1975","unstructured":"Roth G. J., Stanford N., Majerus P. W., Proc. Natl. Acad. Sci. U.S.A.72, 3073 (1975).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/newbio231232a0","volume":"231","author":"Vane J. R.","year":"1971","unstructured":"Vane J. R., Nature New Biol.231, 232 (1971).","journal-title":"Nature New Biol."},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1432-1033.1980.tb04760.x","volume":"109","author":"Van Der Ouderaa F. J.","year":"1980","unstructured":"Van Der Ouderaa F. J., Buytenhek M., Nugteren D. H., Van Dorp D. A., Eur. J. Biochem.109, 1 (1980).","journal-title":"Eur. J. Biochem."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"5192","DOI":"10.1016\/S0021-9258(19)34105-5","volume":"265","author":"DeWitt D. L.","year":"1990","unstructured":"DeWitt D. L., et al., J. Biol. Chem.265, 5192 (1990).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_8_2","first-page":"6610","volume":"268","author":"Meade E. A.","year":"1993","unstructured":"Meade E. A., Smith W. L., DeWitt D. L., ibid268, 6610 (1993).","journal-title":"ibid"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","unstructured":"J. R. Vane and R. M. Botting Scand. J. Rheumatol. 25 (suppl. 102) 9 (1996).","DOI":"10.3109\/03009749609097226"},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1006\/abbi.1995.1130","volume":"317","author":"Bhattacharyya D. K.","year":"1995","unstructured":"Bhattacharyya D. K., Lecomte M., Dunn J., Morgans D. J., Smith W. L., Arch. Biochem. Biophys.317, 19 (1995).","journal-title":"Arch. Biochem. Biophys."},{"key":"e_1_3_1_11_2","unstructured":"Human recombinant COX-2 (5 \u03bcM) in 100 mM tris-HCl buffer (pH 8.0) containing 500 \u03bcM phenol was incubated with several concentrations of APHS or aspirin. Enzyme samples (44 nM) were analyzed periodically for remaining cyclooxygenase activity by treatment with [1- 14 C]arachidonic acid (50 \u03bcM 57 mCi\/mmol) for 30 s at 37\u00b0C. Reactions were terminated by extraction with Et 2 O\/MeOH\/1 M citrate (30:4:1) pH 4.0 (Et ethyl; Me methyl) containing butylated hydroxyanisole (10 \u03bcg) and unlabeled arachidonic acid (10 \u03bcg). The organic phase was spotted on a thin-layer chromatography (TLC) plate (Amersham) and developed in a mobile phase consisting of EtOAc\/CH 2 Cl 2 \/AcOH (75:25:1) at 4\u00b0C. Radiolabeled prostanoids were quantified with a radioactivity scanner (Bioscan Inc. Washington DC). The percentage of total products observed at different inhibitor concentrations was divided by the percentage of total products observed for protein samples preincubated for the same time with dimethyl sulfoxide (DMSO). Semilogarithmic plots of the percent initial enzyme activity remaining versus time were constructed and the apparent rates of pseudo first-order inactivation ( k obs ) at each inhibitor concentration were obtained. The reciprocal values of k obs were plotted against the reciprocal of inhibitor concentration to generate values for k inact and the inhibition constant K i ."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"21438","DOI":"10.1016\/S0021-9258(19)36629-3","volume":"267","author":"Holtzman M. J.","year":"1992","unstructured":"Holtzman M. J., Turk J., Shornick L. P., J. Biol. Chem.267, 21438 (1992).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"1836","DOI":"10.1021\/bi962476u","volume":"36","author":"Xiao G.","year":"1997","unstructured":"Xiao G., et al., Biochemistry36, 1836 (1997).","journal-title":"Biochemistry"},{"key":"e_1_3_1_14_2","unstructured":"[1- 14 C]APHS was synthesized by acetylation of 2-hydroxyphenylhept-2-ynyl sulfide (18 \u03bcmol) with [1- 14 C](CH 3 CO 2 )O (25 \u03bcmol 55 mCi\/mmol) in the presence of dry pyridine (25 \u03bcmol) for 10 hours at room temperature. The crude product was chromatographed on silica gel and eluted with hexanes\/EtOAc (98:2) to afford [1- 14 C]APHS (55 mCi\/mmol) as an oil in 40% yield. Purity was assessed by TLC (hexanes\/EtOAc 90:10; single spot relative mobility \u223c 0.6) and [ 1 H]NMR (CDCl 3 ). For labeling of COX isozymes hematin-reconstituted COX-2 (14.3 \u03bcM) or COX-1 (14.3 \u03bcM) in 100 mM tris-HCl (pH 8.0) containing 500 \u03bcM phenol was treated with 25 equivalents of [1- 14 C]APHS (55 mCi\/mmol) for 2 hours at 25\u00b0C. Radiolabeled proteins were dialyzed at 4\u00b0C for 20 hours. Incorporation of radioactivity into the intact proteins was analyzed by reversed-phase high-performance liquid chromatography (rpHPLC) as follows: The dialyzed proteins were injected on a Vydac C4 column (0.46 \u00d7 25 cm) and eluted with an A-B solvent system [A = 0.1% trifluoroacetic acid (TFA) in water; B = 0.1% TFA in 80% CH 3 CN] and a linear gradient of 40% to 60% CH 3 CN (flow rate = 1 ml\/min). The HPLC system was connected to a Varian 2050 ultraviolet detector (\u03bb = 230 nm) and to a Radiomatic Flo-one \u03b2 radioactive flow detector."},{"key":"e_1_3_1_15_2","unstructured":"[1- 14 C]APHS-labeled COX-2 (1.5 mg) was reduced in 300 mM tris-HCl (pH 8.0) buffer containing 4 M guanidine hydrochloride 2 mM EDTA and 10 mM dithiothreitol for 1.5 hours at 37\u00b0C. Iodoacetamide (25 mM) was added to the reduced protein for 1.3 hours at 25\u00b0C while covered in foil. The protein was extensively dialyzed against 100 mM NH 4 HCO 3 (pH 7.4) and 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and then digested with 44:1 TPCK-trypsin (Sigma) for 21 hours at 37\u00b0C. The digestion was terminated with acid and analyzed by rpHPLC (Beckman ODS C18 column). A single radioactive peak eluted at 21.5 min in a 75-min gradient of 0% to 50% CH 3 CN in 0.1% TFA in water. Further purification of the tryptic digest was effected by rechromatography on a Zorbax SB-C-18 high-resolution column. Analysis of the radiolabeled peptide was conducted by liquid chromatography\u2013mass spectrometry."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"12387","DOI":"10.1016\/S0021-9258(19)49852-9","volume":"267","author":"Shimokawa T.","year":"1992","unstructured":"Shimokawa T., Smith W. L., J. Biol. Chem.267, 12387 (1992).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_17_2","first-page":"2179","volume":"271","author":"Bhattacharyya D. K.","year":"1996","unstructured":"Bhattacharyya D. K., Lecomte M., Rieke C. J., Garavito R. M., Smith W. L., ibid271, 2179 (1996).","journal-title":"ibid"},{"key":"e_1_3_1_18_2","unstructured":"Murine COX-2 mutants were produced using the Stratagene QuickChange site-directed mutagenesis kit. After sequencing to confirm the identity of the mutants the wild-type and mutant murine COX-2 proteins were produced in SF-9 insect cells by means of the pVL1393 expression vector (Pharmingen). The inhibitory potency of APHS against wild-type and mutant murine COX-2 proteins was assessed by incubation of SF-9 cell membranes with APHS or vehicle (DMSO) for 1 hour at 37\u00b0C. The COX reaction was initiated by the addition of [1- 14 C]arachidonic acid (50 \u03bcM) for 1 min at 37\u00b0C. Prostanoid products were measured by TLC (10)."},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/367243a0"},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1038\/nsb1196-927","volume":"3","author":"Luong C.","year":"1996","unstructured":"Luong C., et al., Nature Struct. Biol.3, 927 (1996).","journal-title":"Nature Struct. Biol."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/384644a0","volume":"384","author":"Kurumbail R. G.","year":"1996","unstructured":"Kurumbail R. G., et al., Nature384, 644 (1996).","journal-title":"Nature"},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"15810","DOI":"10.1074\/jbc.271.26.15810","volume":"271","author":"Gierse J. K.","year":"1996","unstructured":"Gierse J. K., et al., J. Biol. Chem.271, 15810 (1996).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_23_2","unstructured":"Q. P. Guo L. H. Wang K. H. Ruan R. J. Kulmacz ibid. p. 19134."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","first-page":"15069","DOI":"10.1073\/pnas.93.26.15069","volume":"93","author":"Landino L. M.","year":"1996","unstructured":"Landino L. M., Crews B. C., Timmons M. D., Morrow J. D., Marnett L. J., Proc. Natl. Acad. Sci. U.S.A.93, 15069 (1996).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_25_2","unstructured":"Low-passage RAW264.7 macrophages were maintained in Dulbecco's modified essential medium (DMEM) + 10% heat-inactivated fetal bovine serum (FBS). Cells (5 \u00d7 10 6 cells per T25 flask) were activated with lipopolysaccharide (500 ng\/ml) and interferon \u03b3 (10 U\/ml) in serum-free DMEM for 7.5 hours then treated with 0 to 2 \u03bcM APHS for 30 min at 37\u00b0C. Exogenous arachidonate metabolism was determined by adding [1- 14 C]arachidonic acid (2 \u03bcM) for 15 min at 25\u00b0C. Experiments were conducted in duplicate and radiolabeled prostanoid products were quantified as in (10)."},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","first-page":"2254","DOI":"10.1172\/JCI119400","volume":"99","author":"Sheng H.","year":"1997","unstructured":"Sheng H., et al., J. Clin. Invest.99, 2254 (1997).","journal-title":"J. Clin. Invest."},{"key":"e_1_3_1_27_2","unstructured":"Single-cell suspensions of HCA-7 Colony 29 cells or HCT-15 cells in cold DMEM + 10% FBS (8 \u00d7 10 4 cells\/ml) were mixed on ice with an equal volume of ice-cold Matrigel matrix (Collaborative Biomedical Products). Cells were seeded into 24-well plates (0.5-ml aliquots) and incubated at 37\u00b0C for 30 min before addition of 1 ml of DMEM + 10% FBS containing DMSO or APHS in DMSO. The cells were treated with 0 to 10 \u03bcM APHS every other day for 14 days at which time the colonies were photographed."},{"key":"e_1_3_1_28_2","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.1016\/0016-5085(94)90246-1","volume":"107","author":"Eberhart C. E.","year":"1994","unstructured":"Eberhart C. E., et al., Gastroenterology107, 1183 (1994).","journal-title":"Gastroenterology"},{"key":"e_1_3_1_29_2","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1172\/JCI116998","volume":"93","author":"DuBois R. N.","year":"1994","unstructured":"DuBois R. N., Awad J., Morrow J., Roberts L. J., Bishop P. R., J. Clin. Invest.93, 493 (1994).","journal-title":"J. Clin. Invest."},{"key":"e_1_3_1_30_2","doi-asserted-by":"crossref","first-page":"3228","DOI":"10.1073\/pnas.91.8.3228","volume":"91","author":"Masferrer J. L.","year":"1994","unstructured":"Masferrer J. L., et al., Proc. Natl. Acad. Sci. U.S.A.91, 3228 (1994).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_31_2","unstructured":"Male Lewis rats (175 g) were injected subcutaneously with 20 ml of sterile air into the intrascapular area of the back. Twenty-four hours later 2 ml of a 1% carrageenan solution in saline was injected into the pouch. Three hours later the test compounds were injected into the pouch as a suspension in 1 ml of 0.5% methylcellulose and 0.025% Tween 20. Three hours after the compound injection the animals were killed and blood was collected for serum thromboxane determination. Pouch fluid was collected and the pouches were washed with 1 ml of saline containing 10 \u03bcM indomethacin. PGE 2 and TxB 2 concentrations were measured by ELISA. Each group contained six animals. No toxicity was observed at APHS doses of 5 or 50 mg\/kg."},{"key":"e_1_3_1_32_2","first-page":"409","volume":"58","author":"Kawamori T.","year":"1998","unstructured":"Kawamori T., Rao C. V., Seibert K., Reddy B. S., Cancer Res.58, 409 (1998).","journal-title":"Cancer Res."},{"key":"e_1_3_1_33_2","unstructured":"For detailed structure-activity relationship studies and synthetic procedures for all compounds see A. S. Kalgutkar et al. in preparation."},{"key":"e_1_3_1_34_2","unstructured":"We thank C. Koboldt for technical assistance H. Sheng for helpful discussions and L. Limbird R. DuBois J. Morrow and J. Staros for critical readings and suggestions. Supported by a research grant and center grants from NIH (CA47479 ES00267 and CA68485). S.W.R. is a recipient of an Australian National Health and Medical Research Council C. J. Martin Fellowship."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.280.5367.1268","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T04:34:33Z","timestamp":1705120473000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.280.5367.1268"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,5,22]]},"references-count":33,"journal-issue":{"issue":"5367","published-print":{"date-parts":[[1998,5,22]]}},"alternative-id":["10.1126\/science.280.5367.1268"],"URL":"https:\/\/doi.org\/10.1126\/science.280.5367.1268","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,5,22]]}}}