Cyclooxygenase-2 Inhibitors in Tumorigenesis (Part I)

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REVIEW Cyclooxygenase-2 Inhibitors in Tumorigenesis (Part I)

Makoto M. Taketo

In Part II of this review, I will summarize earlier data of The rate-limiting enzyme in arachidonate metabolism is me- NSAIDs on colorectal tumors and then present an overview of diated by enzymes known as cyclooxygenases (COXs). These research on COX-2 and its inhibitors relating to cancer. I will enzymes catalyze the biosynthesis of prostaglandin H2, the focus on their roles in colorectal cancer and its animal models, Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021 precursor of molecules, such as prostaglandins, prostacyclin, with some extension to other types of cancer, and discuss their and thromboxanes. The COX enzyme family consists of the clinical relevance. classical COX-1 enzyme, which is constitutively expressed in To understand the effects of COX-2 on cancer, however, it many tissues, and a second enzyme, i.e., COX-2, which is would be essential for us to have a comprehensive knowledge of induced by various stimuli, such as mitogens and cytokines, the biochemistry and pharmacology of the COXs and their in- and is involved in many inflammatory reactions. Because hibitors. I am going to review these studies first in Part I. Be- nonsteroidal anti-inflammatory drugs inhibit both COX-1 cause I am unable to adequately review all of this literature in the and COX-2, these drugs also cause unwanted side effects, space available, I would urge the readers to refer to some ex- exemplified by gastrointestinal bleeding. Accumulating evi- cellent reviews that cover the related topics: Marnett (2), Smith dence indicates that nonsteroidal anti-inflammatory drugs and DeWitt (3,4), DuBois et al. (5), Smith et al. (6), and can reduce the incidence of colorectal cancers in human and Levy (7). This review covers literature available before August experimental animals and can reduce the polyp number and 1997. size in patients with familial adenomatous polyposis. This Part I (of a two-part review) focuses on the discovery of the ARACHIDONATE METABOLISM AND CYCLOOXYGENASES COXs; their biochemical, molecular, and structural properties; and on the discovery of isozyme-specific inhibitors of The enzyme activity that catalyzes formation of prostaglandin COX activity. [J Natl Cancer Inst 1998;90:1529–36] G2 (PGG2) from arachidonic acid—followed by its conversion into PGH2 (Fig. 1)—was designated as COX, prostaglandin– endoperoxide synthase, or prostaglandin H synthase. There are CYCLOOXYGENASES AND CANCER two mammalian isozymes encoded by different genes: the constitutive COX-1 (the first one to be described, EC 1.14.99.1) and More than 20 years ago, high concentrations of prostagland- the inducible COX-2. Both enzymes are the major pharmaco- ins were found in human and animal tumor tissues. This rela- logic targets of NSAIDs. The enzyme we now call COX-1 was tionship of neoplastic tumors to increased levels of prostaglan- first purified and characterized from bull vesicular glands by dins stimulated various experimental and clinical research Miyamoto et al. in 1976 (8), followed by Hemler and Lands (9) thereafter. Accumulating evidence indicates that nonsteroidal from sheep, Van der Ouderaa et al. (10) from sheep, and Ogino anti-inflammatory drugs (NSAIDs) that inhibit prostaglandin (or et al. (11) from bull. In fact, two catalytic reactions take place on prostanoid1) synthesis can reduce the incidence of colorectal the same enzyme: a di-oxygenase activity that cyclizes and oxy- cancers. There are three lines of evidence, from chemical car- genates arachidonic acid to form a15-hydroperoxy-9, 11- cinogen-induced rodent models, from clinical trials of familial endoperoxide with a substituted cyclopentene ring (PGG ), and adenomatous polyposis patients, and from epidemiologic stud- 2 a peroxidase activity that reduces PGG to its 15-hydroxy me- ies. 2 tabolite (PGH ) (8,10,12–15). The approximately 3-kilobase The enzyme involved in the first step of prostanoid synthesis 2 (kb) sheep complementary DNA (cDNA) encoding COX-1 was from arachidonic acid is designated as cyclooxygenase (COX), cloned and analyzed from the seminal vesicles in 1988 (16–18), prostaglandin H synthase, or prostaglandin–endoperoxide syn- followed by cloning from human, mouse, and rat sources (19– thase2. Two isoenzymes exist in the mammalian body, constitutive COX-1 and inducible COX-2. While COX-1 is involved in the homeostasis of various physiologic functions, COX-2 is responsible for many inflammatory processes. After the discov- Affiliation of author: Laboratory of Biomedical Genetics, Graduate School of ery of COX-2 in the early 1990s, many effects of NSAIDs on Pharmaceutical Sciences, University of Tokyo, Japan. Correspondence to: Makoto M. Taketo, M.D., Ph.D., Laboratory of Bio- human colon cancer and in animal models of this disease were medical Genetics, Graduate School of Pharmaceutical Sciences, University of ascribed to these drugs’ effects on COX-2. However, direct ex- Tokyo, 7–3–1 Hongo, Bunkyo, Tokyo 113, Japan (e-mail: [email protected] perimental evidence of this relationship was missing. We re- tokyo.ac.jp). cently presented genetic and pharmacologic data to support the See ‘‘Notes’’ following ‘‘References.’’ hypothesis (1). © Oxford University Press

Journal of the National Cancer Institute, Vol. 90, No. 20, October 21, 1998 REVIEW 1529 repeats (Shaw/Kamen sequences) that are known to mediate rapid and selective mRNA degradation (30,31,33,34). The TIS10 gene was expressed in COS-1 cells and was demonstrated to encode a functional COX containing both hydroperoxidase and COX catalytic activities (35). These data, taken to- gether, established the existence of a novel COX isozyme that was designated as mitogen-inducible prostaglandin G/H synthase (36), prostaglandin H synthase 2, or COX-2. Although the overall exon- intron organization of the mouse and human COX-2 genes is similar to that of human COX-1, the COX-2 genes contain 10 exons, respectively, in about 8 kb, instead of 11 exons in 22 kb for COX-1 (19,24). The relatively small genomic size for the COX-2 gene Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021 fits one of the characteristics of the immediate-early genes (37). Such a difference in the gene structure is reflected in the structural and functional differences Fig. 1. Arachidonate metabolism pathways for prostanoid synthesis by cyclooxygenase 1 between the COX-1 and COX-2 proteins as well (COX-1) and cyclooxygenase 2 (COX-2). (see below). The human COX-2 cDNA was soon cloned from endothelial cells (38,39). Through human–hamster somatic cell hybrid studies, the geno- 22). The amino acid sequences predicted from the nucleic acid mic genes encoding COX-1 and COX-2 were assigned to dif- sequences of the cDNAs are very similar among the species ferent human chromosomes, with the COX-2 gene on (about 90% identity). Sequence comparisons of mouse and chromosome 1 (21,40,41). More precise fluorescence in situ sheep COX-1 suggested that His309 of the sheep enzyme is the hybridization analysis demonstrated that the human genes for axial heme ligand (20), which was confirmed by site-directed COX-1 (PTGS1) and COX-2 (PTGS2) map to 9q32–q33.3 and mutagenesis experiments (23). The genomic sequences for the 1q25.2–q25.3, respectively (42), whereas linkage analyses in human and mouse COX-1 genes span about 22 kb and are com- interspecific backcross mice showed that their mouse homologs posed of 11 exons, respectively (19,24). Ptgs1 and Ptgs2 map to distinct loci on chromosome 2 and Although prostaglandin production and COX-1 messenger chromosome 1, respectively (43). RNA (mRNA) levels were studied extensively with the cDNA probes, most attempts failed to demonstrate a direct association REGULATION OF PROSTAGLANDIN SYNTHESIS (25,26). Rather, analyses using antibodies against COX-1 led to the detection of another cross-reacting band in rat ovary (27) and Before the discovery of COX-2, it was known that prosta- sheep tracheal epithelial cells (28). In 1989, Rosen et al. (29) glandin synthesis is stimulated by a variety of substances, in- found by northern analysis an mRNA band of 4.0 kb in sheep cluding growth factors and tumor promoters. These effects were tracheal mucosa cells that cross-hybridized with the sheep COX thought to be a result of the activation of phospholipases, which cDNA probe under a low stringency condition. The intensity of supply more arachidonic acid to COX (2). The fact that two this band showed a more direct association with prostaglandin isozymes exist that are regulated independently helped clear levels than did the 2.8-kb COX-1 band, and the researchers much of the earlier confusion about the control of prostaglandin suggested that this mRNA encoded a mitogen-inducible COX. production and revealed an elaborate regulatory mechanism for In 1991, cDNAs for COX-2, a novel isozyme of COX, were COX-2. Essentially, COX-1 is expressed constitutively and isolated and sequenced by two independent groups. In chick ubiquitously, whereas COX-2 is expressed only in response to embryo fibroblasts transformed with a temperature-sensitive certain stimuli (3). Rous sarcoma virus (RSV) mutant, Xie et al. (30) found a 4.1-kb mRNA among the immediate early genes that encoded a protein NSAIDS AND THE INHIBITION OF COXS containing about 60% amino acid identity with sheep COX-1. Independently, TIS10, one of seven primary-response genes rap- The early history of aspirin and its mechanism of action has idly and transiently induced by the tumor promoter TPA (12-O- been reviewed in some excellent reviews, especially one by tetradecanoylphobol-13-acetate) in Swiss mouse 3T3 cells, was Vane et al. (44). By the early 1900s, the main therapeutic actions discovered by Kujubu et al. (31) to encode a COX homolog. The of aspirin were known as the drug’s antipyretic, anti- same 4-kb mRNA was identified by O’Banion et al. (32) as a inflammatory, and analgesic effects. In time, several other drugs serum- and glucocorticoid-regulated, COX-related protein, were discovered with similar effects. These are known as either which they cloned and sequenced from C127 mouse fibroblasts ‘‘aspirin-like drugs’’ or ‘‘NSAIDs’’ (Fig. 2) (45). Despite their in 1992. The novel 4-kb mRNA was longer than COX-1 mRNA diverse chemical structures, these drugs share similar therapeutic (2.8 kb), mainly because it contained a much larger 3Ј- properties. They alleviate the swelling, redness, and pain of in- untranslated region of more than 2000 nucleotides. In the 3Ј- flammation; reduce a general fever; and cure a headache. De- untranslated region, researchers found 12–18 copies of AUUUA pending on the dose, they can cause gastric upset, delay the birth

1530 REVIEW Journal of the National Cancer Institute, Vol. 90, No. 20, October 21, 1998 Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021

Fig. 2. NSAIDs (nonsteroidal anti-inflammatory drugs). Reproduced with permission from (45).

process, and even damage the kidneys—not to mention their Table 1. Three classes of NSAIDs based on inhibition kinetics* antithrombotic effect (45). In 1971, Vane (46) reported that aspirin and indomethacin Class I NSAIDs (simple, competitive) Ibuprofen inhibit the biosynthesis of prostaglandins in the guinea pig lung. Mefenamic acid In 1974, [acetyl-3H]aspirin was shown to selectively acetylate a Piroxicam microsomal protein of molecular weight (M ) 85 000 (now Sulindac sulfide (active metabolite of sulindac) r Naproxen known as COX-1) from a sheep and bull seminal vesicles and 6-MNA (active metabolite of nabumetone) from human platelets (47,48). Flower (49) reviewed the pre- Phenylbutazone 1974 data on the inhibitors of prostaglandin biosynthesis. When Flufenamic acid BL-2365 COX-1 was purified in 1976, the enzyme was found to contain Class II NSAIDs (competitive, time-dependent, reversible) both the di-oxygenase and peroxidase activities in a single en- Indomethacin zyme molecule (see above). In the 1980s, numerous enzymatic Flurbiprofen studies were reported that defined the nature of the interactions Meclofenamic acid Diclofenac of various NSAIDs with purified COX-1 (3). It was shown that BL-2338 only the COX activity of COX-1, but not the peroxidase activity, BF 389† was blocked by NSAIDs (50–52). DuP 697† NS-398† Soon after the discovery that COX-2 was the enzyme respon- Class III NSAIDs (competitive, time-dependent, irreversible) sible for inflammatory responses, the effects of various NSAIDs Aspirin (acetylsalicylate acid) on COX-2 were scrutinized and compared with those on COX-1 Valeryl salicylate (53–56). At the same time, rigorous efforts were initiated to search for novel inhibitors that are selective for COX-2 (see *Tabulated from (3,55,57,93,94). below). On the basis of their binding kinetics with the COXs, †Selective COX-2 inhibitors (67,90,95,96). NSAIDs can be grouped into three classes (3). As shown in Table 1, class I compounds are simple, competitive inhibitors that compete reversibly with arachidonic acid for binding to the chains. The best examples of this are BL-2365 and BL-2338, COX active site, whereas class II compounds are competitive, which only require a substitution of a hydrogen for a chlorine time-dependent, and reversible inhibitors. Class III inhibitors— atom to turn class I BL-2365 into class II inhibitor BL-2338 exemplified by aspirin—are known to covalently modify COX-1 (57). and COX-2. Although NSAIDs include a diverse variety of Acetylation of COX-1 by aspirin inactivates the COX activ- structures (as shown in Fig. 2), the difference between the class ity, but not its peroxidase activity (50,51). In contrast, acetyla- I and II compounds are not necessarily in the backbone structure tion of COX-2 by aspirin modifies its COX activity and pro- but are often related to more subtle modifications of the side duces 15R-hydroxyeicosatetraenoic acid (15R-HETE),

Journal of the National Cancer Institute, Vol. 90, No. 20, October 21, 1998 REVIEW 1531 suggesting that aspirin acetylation prevents oxygenation of C- Table 2. IC50 values for NSAIDs in intact cells on COX-1 (bovine 11, resulting in 15R-HETE formation (28,53,58,59). In vitro endothelial cells) and COX-2 (stimulated murine macrophages)* 530 mutagenesis experiments showed that Ser is not required for ␮ IC50 ( M) for the COX activity because its replacement with alanine in sheep Ratio COX-1 yielded a mutant enzyme as active as the wild-type (60). Compound COX-1 (SD) COX-2 (SD) COX-2/COX-1 This and other results suggested that aspirin acetylation of Aspirin 0.3 (0.2) 50 (10) 166 COX-1 prevents arachidonic acid from getting contact with the Indomethacin 0.01 (0.001) 0.6 (0.08) 60 516 Tolfenamic acid 0.0003 (0.0007) 0.005 (0.0019) 16.7 active site. Conversely, replacement of Ser in human COX-2 Ibuprofen 1 (0.07) 15 (5.33) 15 (which corresponds to Ser530 in sheep COX-1) with asparagine Acetaminophen 2.7 (2) 20 (12) 7.4 or alanine did not affect the enzyme activity (58), whereas sub- Sodium salicylate 35 (11.24) 100 (16.2) 2.8 BW755C 0.65 (0.26) 1.2 (0.78) 1.9 stitution with the larger glutamine inactivated the COX (but not Flurbiprofen 0.02 (0.02) 0.025 (0.01) 1.3 the peroxidase) activity. Accordingly, it was suggested that the Carprofen 3 (0.41) 3 (1.72) 1 active site of COX-2 is slightly larger than that of COX-1. This Diclofenac 0.5 (0.21) 0.35 (0.15) 0.7 Naproxen 2.2 (0.98) 1.3 (2.2) 0.6 size difference is also suggested by the broader fatty acid sub- BF 389 0.15 (0.01) 0.03 (0.01) 0.2 strate specificity of COX-2 and the lower relative affinities of Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021

NSAIDs for COX-2 (3,55,61,62). These predictions were con- *Data from (68) with permission. IC50 is the median inhibitory concentration; firmed by crystallographic analysis of COX-1 and COX-2, re- i.e., concentration of the compound that causes half-maximal (50%) inhibition of ס ס spectively (see below). The data obtained with microsomal sus- the enzyme. NSAIDs nonsteroidal anti-inflammatory drugs; SD standard .cyclooxygenase 2 ס cyclooxygenase 1; and COX-2 ס deviation; COX-1 pensions of murine COS-1 cells expressing human COX-1 and COX-2, respectively, are not always consistent with the in vivo anti-inflammatory activity of NSAIDs (e.g., piroxicam or phenylbutazone) (55). Accordingly, it was proposed that the most COX-1 and COX-2. The third group consists of COX-2 selective inclusive approach for determining the NSAIDs biologic activi- inhibitors, the first reports of which were published in 1993. ties is a whole-cell assay involving preincubation with the po- One of the early inhibitors found to have a selectivity for tential inhibitors (55) (see below). COX-2 is BF 389 (Fig. 3, a), an experimental drug with a

Although NSAIDs act principally by inhibition of COX, thus COX-2/COX-1 median inhibitory concentration (IC50) ratio of blocking the initial step in prostanoid synthesis, there appears to 0.2 (67,70). As expected, BF 389 showed little or no gastric be an additional mechanism. Low doses of aspirin and other ulcerogenicity (70). Another compound, NS-398 (Fig. 3, b), was NSAIDs are enough for COX inhibition in vitro and in vivo reported as a novel NSAID that produced minimal stomach le- (46,63), whereas higher doses are required for antirheumatic sions in the rat and that had anti-inflammatory and analgesic effects in vivo (45). In addition, sodium salicylate has minimal effects as potent as those of indomethacin (71). NS-398 was ␮ activity as a COX inhibitor (46), but it is as effective as aspirin found to have an IC50 value of 3.8 M with a purified sheep for inhibiting inflammatory manifestations of arthritis in humans placental COX-2 preparation, whereas sheep vesicular COX-1 and experimental models (45). Recently, Bozza et al. (64) re- was not inhibited even at 0.1 mM (72). NS-398 inhibited only

ported that leukocyte lipid-body formation is inhibited not only excess PGE2 production but did not affect the physiologic level by aspirin, but also by sodium salicylate. Interestingly, this ef- of PGE2 in a rat carrageenan-air pouch inflammation model fect on lipid bodies was independent of COX enzymes because (73,74). it was not impaired in macrophages from homozygous COX-1 In 1994, several more COX-2-selective inhibitors were re-

or COX-2 knockout mice. Accordingly, sodium salicylate and ported. CGP 28232 (Fig. 3, c) showed an IC50 value of 15 nM NSAIDs inhibit archidonic acid-induced lipid-body formation in for COX-2 in interleukin 1-stimulated rat mesangial cells, leukocytes and inhibit the enhanced synthesis of leukotrienes whereas its value for COX-1 in human platelets was 72 ␮M (75).

and prostanoids (64). Conceivably, this inhibition of lipid-body SC-58125 (Fig. 3, d) (76) showed an IC50 value of 50 nM for formation by sodium salicylate and aspirin is mediated by the COX-2 and of greater than 10 ␮M for COX-1 in baculovirus-Sf9 inhibition of NF-␬B (65) and other transcription factors (66). cell homogenates that expressed recombinant mouse enzymes, respectively (77). Moreover, the compound inhibited edema at COX-2 SELECTIVE INHIBITORS AND INHIBITION OF the inflammatory site and was analgesic but had no effect on PG COX ISOENZYMES production in the stomach and caused no gastric toxicity (77). In 1995, L-745 337 (Fig. 3, e) was reported to have an IC50 NSAIDs inhibit the activity of both COX-1 and COX-2, a value of 23 nM for COX-2 in a human osteosarcoma cell line property that accounts for their shared therapeutic and side compared with greater than 10 ␮M for COX-1 in a human my- effects (46). The inhibition of COX-2 may well explain their eloid leukemia cell line (78). The compound effectively inhib- therapeutic use as anti-inflammatory drugs, whereas inhibition ited carrageenan-induced rat paw edema and hyperalgesia but of COX-1 may explain their unwanted side effects, such as did not cause visible gastric legions in rats (78). gastric damage (46). On the basis of the relative inhibition In 1997, a tetrasubstituted furanone, DFU (Fig. 3, f), was of COX-1 versus COX-2 using whole-cell assays, NSAIDs are reported to be a novel, orally active, and highly selective inhibi- classified into three groups (67,68). As shown in Table 2, tor of COX-2 (79). It showed no detectable loss of integrity of aspirin, indomethacin, and ibuprofen are much more potent the gastrointestinal tract, while it effectively inhibited carra- inhibitors of COX-1 than of COX-2. Diclofenac, naproxen, and geenan-induced rat paw edema and hyperalgesia (79). BW 755C (69) are approximately equipotent inhibitors of Before the discovery of COX-2, a novel anti-inflammatory

1532 REVIEW Journal of the National Cancer Institute, Vol. 90, No. 20, October 21, 1998 the COX-2/COX-1 IC50 ratios came out surprisingly different than those for purified enzymes or vesicular systems (82). Most of the compounds were con- sistently more potent against human COX-2 than was observed in earlier data for human COX-2 in microsomal vesicles. However, estimates of inhibitor potency against human COX-1 compare closely with earlier data. The data suggest that human mononuclear cells are exquisitely sensitive to some NSAIDs (82). It is also possible that this discrepancy was a result of various conditions that affect the availability of arachidonic acid and of the allosteric regulation of COX-1 by arachidonic acid and the inhibitors (83). Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021 STRUCTURAL BASIS OF FUNCTIONAL DIFFERENCES BETWEEN COX-1 AND COX-2

In 1994, Picot et al. (84) determined the three- dimensional structure of sheep seminal vesicle COX-1 at 3.5 Å resolution by x-ray crystallography. The enzyme consists of three independent folding units: an epidermal growth factor domain, a membrane-binding motif, and an enzymatic domain. Two adjacent but spatially distinct active sites were found for its heme-dependent peroxidase and COX activities. The COX active site is created by a long hydrophobic channel that is the site of NSAID binding. The conformation of the membrane- binding motif strongly suggests that the enzyme integrates into only one leaflet of the lipid bilayer and is, accordingly, a monotropic membrane protein (85). The COX active site of COX-1 consists of a long, narrow channel (approximately8Å×25Å)extending from the outer surface of the membrane-binding motif to the center (Fig. 4). Tyr385 is located at the BF 389 (97); apex of the channel and sits near the edge of the ס Fig. 3. Structural formulae of cyclooxygenase 2 (COX-2) selective inhibitors. a ס ס ס ס ס b NS-398 (71); c CGP 28 232 (75); d SC-58 125 (76); e L-745 337 (78); f DFU heme plane. Ser530—which is known to be acety- .(FK3311 (81 ס DuP 697 (80); and h ס g ;(98) lated by aspirin—lies just below Tyr385, where its acetylation could easily block access to the upper compound, DuP 697 (Fig. 3, g), was reported that was not (i.e., deeper) part of the channel. ulcerogenic in rats (80). It is a moderate inhibitor of bull seminal The same research group determined the crystal structure of ␮ ס vesicle PG synthesis (i.e., of COX-1 [IC50 24 M]) and a COX-1 that had been inactivated by a potent aspirin analogue, potent inhibitor of rat brain PG synthesis (i.e., of COX-2 [IC50 2-bromoacetoxy-benzoic acid (86). Selective acetylation of ␮M]). DuP 697 turned out later to be highly selective for Ser530 by this analogue abolishes COX-1 activity through steric 4.5 ס COX-2 in whole-cell assays. Another compound, FK3311 (Fig. blockage of the active-site channel, rather than through a large 3, h), was reported to inhibit adjuvant-induced arthritis in rats conformational change. and acetic acid-induced writhing syndrome in mice, without At the end of 1996, two groups reported the crystal structures causing gastrointestinal irritation (81). The compound is also of human and mouse COX-2, respectively, and compared these suggested to be selective for COX-2 based on the human stimu- structures with that of sheep COX-1 (87,88). Luong et al. (87) lated-mononucleocyte assay (see below). determined the crystal structure of COX-2 bound to each of two Using purified enzymes, more precise determinations were selective inhibitors, RS104879 (compound I) and RS57067 reported on the inhibitory effects of COX-2 selective inhibitors. (compound II). The structure of human COX-2 is, in general, At the same time, the discrepancy between the assays with pu- very similar to the sheep COX-1 structure. Amino acid residues rified enzymes and with whole cells became evident, particularly that may be important for enzymatic activity, such as Tyr385 at depending on the species and type of the cells used. For human the COX site and Gln203 at the peroxidase site, are also well platelets and lipopolysaccharide-stimulated mononuclear cells, conserved in the two enzymes. Although the amino acid se-

Journal of the National Cancer Institute, Vol. 90, No. 20, October 21, 1998 REVIEW 1533 Fig. 4. Comparison of the accessible volume of the binding sites in cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) for nonsteroidal anti-inflammatory drugs (NSAIDs). A) representation of the accessible molecular surface at the binding site of flurbiprofen in sheep COX-1 (84). B) Representation of the accessible molecular surface at the NSAID binding site of human COX-2, with compound I (RS 104879) bound (87). Reproduced from Nat Struct Biol 1996;3: 927–33 with permission from the authors and the copyright holder. Downloaded from https://academic.oup.com/jnci/article/90/20/1529/2519830 by guest on 27 September 2021 quence of the membrane domain is the least conserved between COX active site. The binding site of flurbiprofen in COX-2 is COX-1 and COX-2, exhibiting only 33% identity, the secondary identical to that observed in COX-1.3 and tertiary structures of this domain are well conserved. The single amino acid insertion at position 106 in COX-2 (106a) REFERENCES does not perturb the overall structure (87). The only residue lining the channel that is not identical be- (1) Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, tween COX-1 and COX-2 is amino acid residue 523, which is Ile et al. Suppression of intestinal polyposis in Apc⌬716 knockout mice by in COX-1 and Val in COX-2. This difference has consequences inhibition of cyclooxygenase 2 (COX-2) Cell 1996;87:803–9. for the size and shape of the NSAID binding site. The COX-2 (2) Marnett LJ. Aspirin and the potential role of prostaglandin in colon cancer. structure also contains a second internal pocket extending off the Cancer Res 1992;52:5575–89. (3) Smith WL, DeWitt DL. Biochemistry of prostaglandin endoperoxide H NSAID binding site. 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