Multiple Sites on Prostaglandin Cyclooxygenase Are

Proc. Nati. Acad. Sci. USA Vol. 78, No. 4, pp. 2053-2056, April 1981 Biochemistry

Multiple sites on prostaglandin cyclooxygenase are determinants in the action of nonsteroidal antiinflammatory agents (supplementary site/catalytic site/ulcers/arachidonic acid oxygenation) J. L. HUMES, C. A. WINTER, S. J. SADOWSKI, AND F. A. KUEHL, JR. Department of Biochemistry of Inflammation, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 Communicated by JosefFried, December 8, 1980

ABSTRACT Evidence is presented to show that nonsteroidal of these laboratories. Arachidonic acid was purchased from antiinflammatory drugs react with two sites on the cyclooxygenase Sigma. Other materials were obtained from standard suppliers. (8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, Microsomes containing cyclooxygenase activity were pre- EC 1.14.99.1). Although the degree of interaction with the cata- pared from ram seminal vesicles as described (7). The NSAIDs lytic site determines the potency of such compounds, interaction were incubated for 2 min at 30'C with approximately 3 mg of with the supplementary site is also obligatory for efficacy as cy- microsomal protein in 3 ml of0.1 M potassium phosphate buffer clooxygenase inhibitors and may explain the selectivity of such (pH 7.6) in a Yellow Springs Instrument Co. model 53 oxygen drugs in inhibiting the cyclooxygenase but not the lipoxygenase monitor. The reactions were initiated by the addition ofarach- pathway. Drugs that interact more effectively with the supple- idonic acid to a final concentration of The rate mentary site than with the catalytic site-i.e., those of weak to 110 AM. ofoxygen moderate activity as cyclooxygenase inhibitors-are shown to pre- consumed from the reaction mixture was determined from the vent inhibition ofthe enzyme by indomethacin. Compounds in this initial linear portion ofthe curve. Dimethyl sulfoxide was used class are also capable of blocking the ulcerogenic action of indo- as a solvent for both the NSAID and the arachidonic acid. The methacin, which suggests that this antiulcerogenic property stems total amount added to the monitor chambers did not exceed 100 from a direct action at the level of the cyclooxygenase in the p.l; this amount of dimethyl sulfoxide showed no effect on ox- stomach. ygen incorporation. Protein was determined by the method of Murphy and Kies (8). There are a number of reports indicating that the ulcerogenic For some studies, the cyclooxygenase activity was solubilized action of high doses of nonsteroidal antiinflammatory drugs by stirring the microsomes (75 mg of protein) under nitrogen (NSAIDs) is secondary to a depression of prostaglandin (PG) for 30 min at 40C in 3 ml of 2% (wt/vol) S10-7 detergent in 0.1 synthesis in the stomach (1, 2). The most convincing evidence M potassium phosphate buffer (pH 7.6). The mixture was cen- in support of this concept is the finding that coadministration trifuged for 2 hr at 105,000 x g and the supernatant phase was of PGs prevents induction of lesions by such drugs (2). Oral used as the source of cyclooxygenase. administration of salicylic acid-a weak inhibitor of cyclooxy- Protection Against Indomethacin-Induced Gastric Hemor- genase (8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxi- rhages. Male Sprague-Dawley rats weighing 125-150 g were doreductase, EC 1.14.99.1), the initial step in PG biosynthesis fasted overnight. The NSAIDs were suspended in 5% (wt/vol) and the target of most NSAIDs-has been found to diminish methyl cellulose and administered orally. The rats were killed gastric lesions resulting from high levels of indomethacin and 4 hr later with pentobarbital and the stomachs were removed. other NSAIDs in rats (3). A similar action of 5-(2,4-difluoro- Any hemorrhagic area on the stomach 2 mm or greater in its phenyl)-salicylic acid (diflunisal), an inhibitor ofcyclooxygenase largest dimension was considered positive. of intermediate potency, has been reported (4). In view of the requirement for endogenous synthesis of PGs for homeostasis RESULTS ofthe stomach, these findings with both salicylate and diflunisal could be due to their ability, after oral administration, to pre- Cyclooxygenase Inhibition. Salicylic acid at a level that has vent the local transport of indomethacin to the active site on no detectable effect upon cyclooxygenase activity (2000 A.M) stomach cyclooxygenase or due to direct competition with in- shifted the ID50 (mean infective dose) ofindomethacin from 0.16 domethacin for a common site on the enzyme. The recent find- to 1.1 A.M (Fig. 1). In a similar manner, 0.8 p.M diflunisal, also ing that salicylic acid also blunts the ulcerogenic action of in- a level that does not inhibit the cyclooxygenase, shifted the indomethacin when administered parenterally may be interpreted domethacin ID50 to 0.7 p.M. These data suggest that salicylate to support this latter possibility (5). To explore this question and undergoes an interaction at a locus on the enzyme with an af- its possible relation to ulcerogenicity, a number of NSAIDs finity that is 3 orders ofmagnitude less than that for indometh- were examined for their ability to counter the effect of indo- acin, whereas diflunisal has an affinity for this site approximately methacin in inhibiting cyclooxygenase activity in vitro and to equivalent to that of indomethacin. suppress indomethacin-induced gastric lesions in vivo. Diflunisal at a concentration of 0.8 p.M shifted the ID50 for aspirin from 50 to a value of350 p.M (Fig. 2). In a similar manner, salicylate, at a substantially higher level (2000 A.M), shifted MATERIALS AND METHODS the ID50 to 850 p.M. These data suggest that aspirin interacts PG Cyclooxygenase. The NSAIDs and surfactant S10-7 (6) at a site on the cyclooxygenase, common to salicylate and di- were obtained from N. Jensen and R. W. Egan, respectively, flunisal. Either this site is a target for the acetylation or its occupancy is obligatory for the acetylation reaction to occur. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be herebv marked "advertise- Abbreviations: NSAID(s), nonsteroidal antiinflammatory drug(s); PG(s), ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. prostaglandin(s); ID50, mean infective dose. 2053 Downloaded by guest on September 29, 2021 et 2054 Biochemistry: Humes al. Proc. Natl. Acad. Sci - USA 78 (1981)

100 100 80 80 0 ._ 0 ._'. ._ 60 ,,0 , 0 ._4 60 _ 0/10

._ *e- 40 40 _ 20

20 _ 0 I I IIII aI 0 30 60 90 120 300

1-8 jo-7 10-6 Time, sec Indomethacin, M FIG. 3. Time-dependent indomethacin inhibition of PG cyclooxy- FIG. 1. Diflunisal and salicylic acid prevent indomethacin inhi- genase in the presence and absence of diflunisal. Indomethacin alone bition of PG cyclooxygenase. NSAIDs were incubated for 2 min with or indomethacin with diflunisal were incubated with ram vesicular ram vesicular gland microsomes. The reaction was initiated by the gland microsomes for various periods of time. The reaction was initi- addition ofarachidonic acid to a final concentration of 110 AM, and the ated by the addition of arachidonic acid to a final concentration of 110 rate of oxygen consumed from the reaction mixture was determined. AuM, and the rate of oxygen consumed from the reaction mixture was The reaction rate was 475 ± 11 nmol (oxygen) min- mg-' (protein). determined. o-o, Indomethacin (0.3 AM) alone; *, indometh- o-o, Indomethacin alone; x-x, indomethacin and 0.8 ,uM di- acin (0.3 AM) and diflunisal (0.8 AM). flunisal; o---co indomethacin and 2000 AuM salicylic acid. to moderate activity, as represented by salicylic acid, acetamin- Indomethacin equilibrated with the cyclooxygenase well ophen, and diflunisal, occurred in a dose-related manner and within the 2-min time frame employed in these studies (Fig. paralleled the efficacy of these same compounds in preventing 3). The addition of diflunisal, which unlike indomethacin has indomethacin-induced ulcers in rats. This correlation was ex- no discernible time requirement for inhibition, lowered the tended to potent cyclooxygenase inhibitors, as represented by efficacy of indomethacin as a cyclooxygenase inhibitor prior to ketoprofen, which was additive to the action of indomethacin and subsequent to the time interval required for indomethacin in both inhibiting cyclooxygenase and inducing ulcers. This re- to elicit full inhibitory activity. lationship between these parameters makes it possible to sug- In order to preclude the possibility that diflunisal might com- gest that the property of salicylate and related compounds to pete for the transport of indomethacin to the active site on the blunt the ulcerogenic action of indomethacin depends upon enzyme in the microsomal particle, the effect of diflunisal on unique interaction at the level of the cyclooxygenase. indomethacin inhibition was evaluated in a surfactant-solubilized cyclooxygenase preparation. As with the microsomal enzyme, diflunisal effectively prevented the inhibitory action of DISCUSSION indomethacin on surfactant-solubilized enzyme (Table 1). The finding that salicylic acid and its structural relative diflunisal The relevance of the interactions of the NSAIDs at the level diminish the efficacy ofindomethacin as an inhibitor ofcycloox- of the cyclooxygenase to their actions suppressing indometha- ygenase fosters the concept that competition between the cin-induced ulcers is apparent from the data in Table 2. The NSAIDs occurs either in gaining access to the enzyme or at the ability to blunt cyclooxygenase inhibition by NSAIDs of weak level of the enzyme itself. The former possibility was excluded by studies with surfactant-solubilized enzyme in which a similar competitive interaction was obtained. This soluble enzyme

100 0X preparation does not represent micellar aggregates of the enzyme, as the cyclooxygenase activity was purified to homo- geneity from it (R. W. Egan, E. Baptista, and P. Gale, personal communication). Thus, it is evident that competition occurs on

60- Table 1. Diflunisal prevents indomethacin inhibition of detergent-solubilized PG cyclooxygenase 40 02 consumed, 20 le Concentration, nmol Inhibition, ILM min-'mg-' %

- 4 0 10-2 No additions 318 10-6 10-5 104 10-3 1 Diflunisal 83 344 + 5 0 Aspirin, M Indomethacin 0.3 87 1 73 Diflunisal with FIG. 2. Diflunisal and salicylic acid prevent aspirin inhibition of indomethacin 83 + 0.3 334 11 0 PG cyclooxygenase. Oxygen consumption catalyzed by ram vesicular gland microsomes was measured as described in Fig. 1. o-o, As- PG cyclooxygenase was solubilized from ram vesicular gland micro- - were incubated as described. The data are the pirin; x x, aspirin and 0.8 jxM diflunisal; > ---, aspirin and 2000 somes, and the NSAIDs ;LM salicylic acid. mean of three experiments ± SD. Downloaded by guest on September 29, 2021 Biochemistry: Humes et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2055

Table 2. Some nonsteroidal antiinflammatory agents prevent indomethacin inhibition of PG cyclooxygenase and protect rats from.indomethacin induced gastric hemorrhages In vitro PG cyclooxygenase In vivo stomach lesions Concentration, Inhibition, Dose, Molar Lesions, NSAID AM % mg/kg ratio no. Indomethacin alone 0.16 53 20 - 4.6 ± 0.5 (34) Indomethacin with: Salicylic acid 200 37 72 8 2.2 ± 0.7 (6) (ID5o > 5000 /LM) 330 24 144 16 2.0 ± 1.1 (6) 660 0 288 32 0.6 ± 0.2 (12) Acetaminophen 20 58 3.8 0.45 3.4 ± 0.7 (9) (ID50= 5000 AM) 67 18 38 4.5 3.1 ± 0.5 (9) 200 0 190 22.5 0.8 ± 0.5 (9) Diflunisal 8 0 56 4 2.0 ± 0.7 (6) (ID50 = 25 ALM) 112 8 1.3 ± 0.9 (6) 224 16 0.8 ± 0.5 (12) Ketoprofen 0.08 88 28 2 3.4 ± 0.7 (8) (ID50 = 0.6 uM) 142 10 8.4 ± 0.8 (8) PG Cyclooxygenase. A concentration of indomethacin (0.16 ,uM) to elicit 50% inhibition of the incorporation of oxygen was incubated with various NSAIDs. The data from three experiments are shown. The enzyme activity ranged from 580-790 nmol (ofoxygen) min-' mg-' (protein). The ID50 ofthe various NSAIDs as cyclooxygenase inhibitors were determined as described, except that 500 AM phenol was included in the incubation mixtures. Acetaminophen, 4'-hydroxyacetanilide; diflunisal, 5-(2,4- difluorophenyl)-salicylic acid; ketoprofen, 2-(3-benzoylphenyl)propionic acid. Stomach lesions. Indomethacin, (20 mg/kg), was administered orally to fasted rats. The NSAIDs tested for their protective effect were given in a physical mixture with indomethacin. The molar ratio is [NSAID]/[indomethacin]. The data are expressed as the mean ± SEM. The number ofrats in each study is shown in parentheses. the enzyme itself. However, because these interactions take cussed, the sulfoxide is a compound with a weak affinity for the place at levels of salicylic acid and diflunisal that do not inhibit supplementary site but a high affinity for the catalytic site, the oxygenation of substrate arachidonic acid, competition at whereas its highly lipophilic reduced counterpart interacts ef- the catalytic site is excluded. These data require the existence ficiently with both sites to yield a cyclooxygenase inhibitor ofat least two distinct but interacting sites on the cyclooxygen- equivalent in potency to indomethacin. ase with which the NSAIDs may react: the catalytic site and a The inhibition of cyclooxygenase by indomethacin has been supplementary one. In one possible model, occupancy at the shown to be a time-dependent process (10). The equilibrium supplementary site alters the catalytic site in such a manner that between indomethacin and the enzyme was essentially com- indomethacin is no longer able to bind to it. However, because pleted within 60 sec, as shown by the maximal inhibitory effects. indomethacin is a competitive inhibitor of arachidonic acid such Diflunisal does not alter the time-dependence of the indo- an allosteric alteration of the catalytic site by the salicylates methacin inhibition; however, diflunisal suppressed the indo- might be expected to result also in their inhibition of cycloox- methacin inhibition at all times studied. Thus, the 2-min in- ygenase activity, a phenomenon not observed at the levels em- cubation employed in these studies allowed complete equilibrium ployed. Thus, the preferred model for the multiple-site thesis between the cyclooxygenase and the NSAIDs. Unlike indo- is one in which interaction with the supplementary site facili- methacin, the sulfide (cis-5-fluoro-2-methyl-1-[p-(methylthio)- tates the action ofa given NSAID as a cyclooxygenase inhibitor. benzylidene]indene-3-acetic acid, is not a time-dependent in- Additional facts bearing on the nature ofthe catalytic site are hibitor of the cyclooxygenase; however, like indomethacin its consistent also with the preferred model. Introduction of the efficacy as a cyclooxygenase inhibitor is blunted by diflunisal highly lipophilic difluorophenyl group para to the phenolic (data not shown). These data suggest that interactions between moiety ofsalicylic acid increases its potency as a cyclooxygenase weak and potent NSAIDs may be a general phenomenon. inhibitor from a nondetectable value (ID50 > 2000 ,uM) to 25 Aspirin has been shown to inhibit PG biosynthesis by acety- MuM and increases its efficacy in blunting the action of indo- lating an amino acid that is critical to the action of cyclooxy- methacin by 1000-fold. These data suggest that the supple- genase (11). This acetylation is prevented by indomethacin, mentary site is a highly lipophilic area of the enzyme, whose diflunisal, and substrate arachidonic acid (11, 12). The finding occupancy is essential for potency as an NSAID. Thus, the ef- in this report that salicylate and diflunisal impede the inhibitory ficacy ofdiflunisal in blunting the action ofindomethacin would action of aspirin requires that they prevent this acetylation re- result from a high affinity for this site and a lower affinity for the action. Because substrate arachidonic acid has been shown to catalytic site as compared with the more potent NSAID. More inhibit this transacetylation, data in this report indicating that dramatic evidence ofthe requirement for a lipophilic site is seen salicylic acid and diflunisal at the levels employed do not com- in the action ofcis-5-fluoro-2-methyl-1-[p-(methylsulfinyl)-ben- pete with arachidonic acid permits the suggestion that aspirin zylidene]indene-3-acetic acid. This compound is essentially also interacts at the supplementary site, an action that facilitates devoid ofactivity as a cyclooxygenase inhibitor (ID50, 290 LM). the acetylation at the locus of the catalytic site. However, when the sulfoxide group is reduced to the sulfide- The observation that acetaminophen (Table 2), a weak cy- a reaction resulting in a 1000-fold increase in lipid solubility, clooxygenase inhibitor, also is capable ofblunting the inhibitory as reflected by its distribution between ether/water-efficacy effect of indomethacin indicates that such interactions by as a cyclooxygenase inhibitor is increased 3 orders ofmagnitude NSAIDs ofweak to moderate potency are not restricted to sal- to an ID50 of0.1 uMM, a value equivalent to that for indomethacin icylic acid and its structural relatives. Competition is not evi- itself (9). Thus, in accord with the principles previously dis- dent for the potent inhibitor, ketoprofen. In this case, additivity Downloaded by guest on September 29, 2021 2056 Biochemistry: Humes et al. Proc. Natl. Acad. Sc'i. USA 78 (1981) is noted. The correlation between the properties of the weaker by soybean lipoxygenase is not sensitive to indomethacin (20). NSAIDs to blunt indomethacin inhibition ofthe cyclooxygenase The initial reaction of arachidonic acid with both cycloxy- and the additive effect of the potent inhibitor ketoprofen pre- genase and soybean lipoxygenase has been shown to involve the cisely parallels their actions on indomethacin-induced ulcers. abstraction ofa hydrogen atom at C-13 ofthe acid to form a rad- This degree ofcorrelation fosters the conclusion that the prop- ical intermediate (21, 22). Competitive inhibitors ofarachidonic erty of these compounds to blunt the ulcerogenic effect of in- acid would be expected to act at this step and, thus, inhibit both domethacin stems from an action at the level of the cyclooxy- the cyclooxygenase and lipoxygenase. 5,8,11, 14-Eicosatetray- genase. There are other reports that weak NSAIDs block the noic acid is an inhibitor of both enzymes; however, it is not an actions of other such drugs. Salicylic acid has been shown to effective antiinflammatory agent because of its ineffectual reduce the efficacy of aspirin as an inhibitor of both rabbit and suppression ofcyclooxygenase at the inflamed joint. In contrast, human platelet aggregation (13, 14). Sulfinpyrazone interferes the unique efficacy of indomethacin and other NSAIDs can be with aspirin inhibition of serotonin release from platelets (15). explained by their Ki values, which are lower than the Km value Salicylic acid also reduces the protective effect ofaspirin against for substrate arachidonic acid by 2 orders of magnitude. In ef- bronchoconstrictive and hypotensive actions induced by ad- fect, such NSAIDs have far greater affinity for the cyclooxy- ministered arachidonic acid in guinea pigs and rabbits (16). genase than for its natural substrate, a fact that makes their ac- Despite the fact that enzymatic activity alone was determined tion so consequential. We suggest that the unique potency and in these studies, implicit in the interactions of NSAIDs with specificity of NSAIDs in their action on the cyclooxygenase may cyclooxygenase is the involvement of distinct binding sites on be attributable to the presence of the putative supplementary the enzyme; binding is considered to be obligatory in compet- site on the cyclooxygenase and its absence in the lipoxygenase. itively inhibiting such enzymatic conversions. In an effort to obtain direct evidence of such binding phenomenon, the ability of relevant compounds to displace radioactive diflunisal was 1. Robert, A. (1974) Gastroenterology 66, 765. examined with the solubilized enzyme (R. W. Egan, E. Bap- 2. Robert, A. (1976) in Advances in Prostaglandin and Thrombox- tista, and F. A. Kuehl, Jr., unpublished data). However, the ane Research, eds. Samuelsson, B. & Paoletti, R. (Raven, New degree of nonspecific binding to the protein was of such a mag- York), Vol. 2, pp. 507-520. 3. Ezer, E., Palosi, E., Haj6s, G. & Szporny, L. (1976) J. Pharm. nitude that competitive binding studies were not possible. This Pharmacol. 28, 655-656. is not surprising in view of the fact that avid binding of NSAIDs 4. Conti, P. & Continenza, A. (1980)J. Pathol. 131, 357-362. to proteins is a requisite for effective antiinflammatory activity 5. Corell, T. & Jensen, K. M. (1979) Acta Pharmacol. Toxicol. 45, (17), and a high degree ofcorrelation between binding to serum 225-231. albumin and efficacy of NSAIDs has been reported (18). Such 6. Egan, R. W., Jones, M. A. & Lehninger, A. L. (1976) J. Biol. lipophilic pockets are present in albumin, evident by its ability Chem. 251, 4442-4447. 7. Egan, R. W., Paxton, J. & Kuehl, F. A., Jr. (1976)J. Biol. Chem. to bind fatty acids. Similar lipophilic sites likely occur in other 251, 7329-7335. proteins. The ubiquitousness of these lipophilic peptide se- 8. Murphy, J. B. & Kies, M. W. (1960) Biochim. Biophys. Acta 45, quences may explain the ability of many NSAIDs, including 382-384. indomethacin, to inhibit a plethora of enzymes at levels several 9. Kuehl, F. A., Jr., Dougherty, H. W., Ham, E. A., Humes, J. L., orders of magnitude greater than that required to inhibit cy- Egan, R. W. & Winter, C. A. (1980) in Current Concepts on clooxygenase and may have little bearing on the structural fea- Anti-Inflammatory Drugs, eds. Miehlke, K., Otte, P. & Platz, C. M. (Biomedical Information, New York), pp. 17-31. tures of such drugs that interact with the catalytic site of the 10. Rome, L. H. & Lands, W. E. M. (1975) Proc. Natl. Acad. Sci. cyclooxygenase. USA 72, 4863-4865. The reaction of arachidonic acid with soybean lipoxygenase 11. Roth, G. J., Stanford, N. & Majerus, P. W. (1975) Proc. Natl. results in the introduction of a hydroperoxy group at carbon 15 Acad. Sci. USA 72, 3073-3076. (C-15). In contrast, the first oxygenation ofarachidonic acid with 12. Majerus, P. W. & Stanford, N. (1977) Br.J. Clin. Pharmacol .4, cyclooxygenase results in the formation of a peroxy radical 15S-18S. PG 13. Vargaftig, B. B. (1978) Eur. J. Pharmacol. 50, 231-241. at C-ll, followed by isomerization and the introduction of a 14. Merino, J., Livio, M., Rajtar, G. & deGaetano, G. (1980) hydroperoxy group at C-15 to yield PGG2. In the absence ofthe Biochem. Pharmacol. 29, 1093-1096. C-8 double bond in an eicosanoic acid, cyclization and intro- 15. Ali, M. & McDonald, J. W. D. (1979) Prostaglandins & Med. 3, duction of a second molecule of oxygen is not possible. How- 327-332. ever, such C-8 unsaturation is not essential to the initial reaction 16. Lefort, J. & Vargaftig, B. B. (1978) Br.J. Pharmacol. 63, 35-42. with the enzyme because a acid, 17. Graf, P., Glatt, M. & Brune, K. (1975) Experientia 31, 951-953. w-6-dienoic 11,14-eicosadi- 18. Gryglewski, R. (1974) in Prostaglandinrr Synthetase Inhibitors, enoic acid, is a substrate for the cyclooxygenase (19). The prod- eds. Robinson, H. J. & Vane, J. R. (Raven, New York), pp. uct of this oxidation is the ll-hydroperoxy acid. Additional evi- 32-52. dence that the w-6 diene is the major determinant in the initial 19. Hemler, M. E., Crawford, C. G. & Lands, W. E. M. (1978) Bio- reaction with the cyclooxygenase is shown by our observation chemistry 17, 1772-1779. that the 18-carbon, w-6 diene fatty acid linoleic acid is also a 20. Downing, D. T., Ahern, D. G. & Bachta, M. (1970) Biochem. substrate for the cyclooxygenase, yielding the Biophys. Res. Commun. 40, 218-223. corresponding 9- 21. Samuelsson, B. (1972) Fed. Proc. Fed. Am. Soc. Exp. Biol. 31, hydroperoxy acid (data not shown). In each of these cyclooxy- 1442-1450. genase-mediated lipoxygenase reactions, indomethacin is'a po- 22. Beerthuis, R. K., Nugteren, D. H., Pabon, H. J. J. & Van Dorp, tent inhibitor. Conversely, oxygenation of such w-6 diene acids D. A. (1968) Recl. Trav. Chim. Pays Bas 87, 461480. Downloaded by guest on September 29, 2021