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• W2000748742 abstract "Lumiracoxib is the first example of a marketed COX-2 inhibitor of the arylacetic acid class, and it is reported to be the most selective COXIB in vivo. However, the molecular basis of its COX-2 inhibition has not been completely defined. Using standard assays, lumiracoxib was found to be a poor inhibitor of purified ovine COX-1 and a relatively weak inhibitor of purified human COX-2. The extent of COX-2 inhibition plateaued at around 50% and suggested that the inhibitor may be reversibly bound to the enzyme. Kinetic studies with lumiracoxib demonstrated that it was a time-dependent and slowly reversible inhibitor of human COX-2 that exhibited at least two binding steps during inhibition. Derivatives of lumiracoxib were synthesized with or without the methyl group on the phenylacetic acid ring and with various substitutions on the lower aniline ring. Inhibition studies demonstrated that the methyl group on the phenylacetic acid ring is required for COX-2 selectivity. The chemical identity and position of the substituents on the lower aniline ring were important in determining the potency and extent of COX inhibition as well as COX-2 selectivity. Mutation of Ser-530 to Ala or Val-349 to Ala or Leu abolished the potent inhibition observed with wild-type human COX-2 and key lumiracoxib analogs. Interestingly, a Val-349 to Ile mutant was inhibited with equal potency to human COX-2 with 2,6-dichloro-, 2,6-dimethyl-, or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Taken together with a recent crystal structure of a lumiracoxib-COX-2 complex, the kinetic analyses presented herein of the inhibition of mutant COX-2s by lumiracoxib allows the definition of the molecular basis of COX-2 inhibition. Lumiracoxib is the first example of a marketed COX-2 inhibitor of the arylacetic acid class, and it is reported to be the most selective COXIB in vivo. However, the molecular basis of its COX-2 inhibition has not been completely defined. Using standard assays, lumiracoxib was found to be a poor inhibitor of purified ovine COX-1 and a relatively weak inhibitor of purified human COX-2. The extent of COX-2 inhibition plateaued at around 50% and suggested that the inhibitor may be reversibly bound to the enzyme. Kinetic studies with lumiracoxib demonstrated that it was a time-dependent and slowly reversible inhibitor of human COX-2 that exhibited at least two binding steps during inhibition. Derivatives of lumiracoxib were synthesized with or without the methyl group on the phenylacetic acid ring and with various substitutions on the lower aniline ring. Inhibition studies demonstrated that the methyl group on the phenylacetic acid ring is required for COX-2 selectivity. The chemical identity and position of the substituents on the lower aniline ring were important in determining the potency and extent of COX inhibition as well as COX-2 selectivity. Mutation of Ser-530 to Ala or Val-349 to Ala or Leu abolished the potent inhibition observed with wild-type human COX-2 and key lumiracoxib analogs. Interestingly, a Val-349 to Ile mutant was inhibited with equal potency to human COX-2 with 2,6-dichloro-, 2,6-dimethyl-, or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Taken together with a recent crystal structure of a lumiracoxib-COX-2 complex, the kinetic analyses presented herein of the inhibition of mutant COX-2s by lumiracoxib allows the definition of the molecular basis of COX-2 inhibition. The cyclooxygenase (COX) 2The abbreviations used are: COX, cyclooxygenase; mCOX, oCOX, hCOX, and m/hCOX, murine, ovine, human, and murine and human COX, respectively; AA, arachidonic acid; NSAID, nonsteroidal anti-inflammatory drug; SAR, structure activity relationship; HPLC, high pressure liquid chromatography.2The abbreviations used are: COX, cyclooxygenase; mCOX, oCOX, hCOX, and m/hCOX, murine, ovine, human, and murine and human COX, respectively; AA, arachidonic acid; NSAID, nonsteroidal anti-inflammatory drug; SAR, structure activity relationship; HPLC, high pressure liquid chromatography. enzymes COX-1 and COX-2 share high sequence homology (60%), have very similar three-dimensional structures, and catalyze the conversion of arachidonic acid (AA) to prostaglandin H2. Lumiracoxib is a highly selective COX-2 inhibitor that is weakly acidic and displays a unique pharmacological profile that includes rapid absorbance and a relatively short plasma half-life (1.Lyseng-Williamson K.A. Curran M.P. Drugs. 2004; 64: 2237-2246Crossref PubMed Scopus (19) Google Scholar). Lumiracoxib displays a 500-fold greater selectivity for COX-2 than COX-1 in vivo (2.Tannenbaum H. Drugs. 2004; 64: 2247-2248Crossref Scopus (3) Google Scholar) and in clinical studies has shown a 3–4-fold reduction in ulcer complications versus classical NSAIDs (3.Farkouh M.E. Kirshner H. Harrington R.A. Ruland S. Verheugt F.W. Schnitzer T.J. Burmester G.R. Mysler E. Hochberg M.C. Doherty M. Ehrsam E. Gitton X. Krammer G. Mellein B. Gimona A. Matchaba P. Hawkey C.J. Chesebro J.H. Lancet. 2004; 364: 675-684Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 4.Schnitzer T.J. Burmester G.R. Mysler E. Hochberg M.C. Doherty M. Ehrsam E. Gitton X. Krammer G. Mellein B. Matchaba P. Gimona A. Hawkey C.J. Lancet. 2004; 364: 665-674Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). Lumiracoxib lacks the tricyclic structure of the diarylheterocycle class of COX-2-selective inhibitors (e.g. celecoxib and rofecoxib) and does not contain a sulfonamide or sulfone group. Instead, lumiracoxib is a close structural analog of diclofenac (Fig. 1). Although lumiracoxib and diclofenac share structural similarities, they exhibit large differences in the selectivity of COX-2 inhibition. The molecular basis for this difference in the selectivity of COX inhibition is not entirely understood. Previous crystal structures of COX enzymes with carboxylic acid-containing NSAIDs reveal that the inhibitors are typically positioned with their carboxylates coordinated to Arg-120 and their aromatic functional groups projecting up into the cyclooxygenase active site (5.Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1145) Google Scholar, 6.Kurumbail 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. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1578) Google Scholar). However, a crystal structure of diclofenac bound in the active site of COX-2 (Fig. 2B) revealed an inverted binding mode of the molecule with its carboxylic acid moiety coordinated to Ser-530 and Tyr-385 (7.Rowlinson S.W. Kiefer J.R. Prusakiewicz J.J. Pawlitz J.L. Kozak K.R. Kalgutkar A.S. Stallings W.C. Kurumbail R.G. Marnett L.J. J. Biol. Chem. 2003; 278: 45763-45769Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Structure-activity studies have found that another distinctive binding mode is exhibited by indomethacin, a nonselective NSAID that is a time-dependent, functionally irreversible inhibitor of COX enzymes (8.Prusakiewicz J.J. Felts A.S. Mackenzie B.S. Marnett L.J. Biochemistry. 2004; 43: 15439-15445Crossref PubMed Scopus (49) Google Scholar). Indomethacin binds in the cyclooxygenase active site with its carboxylic acid group ion-pairing and hydrogenboding with Arg-120 and Tyr-355 (Fig. 2A). In addition, its 2′-methyl group binds in a hydrophobic pocket composed of Ala-527, Val-349, Ser-530, and Leu-531 (6.Kurumbail 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. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1578) Google Scholar). Mutations of Val-349 in this methyl-binding pocket to alanine or leucine alter the size of the pocket and lead to an increase or decrease in the potency of indomethacin, respectively. The 2′-des-methyl analog of indomethacin does not inhibit COX-1 and is a very weak, rapidly reversible inhibitor of COX-2 (8.Prusakiewicz J.J. Felts A.S. Mackenzie B.S. Marnett L.J. Biochemistry. 2004; 43: 15439-15445Crossref PubMed Scopus (49) Google Scholar). Recently, the crystal structure of murine COX-2 with lumiracoxib was solved and revealed that lumiracoxib binds in an inverted orientation similar to that of diclofenac (9.Clark K. Kulathila R. Koehn J. Rieffel S. Strauss A. Hu S. Kalfoglou M. Szeto D. Lasala D. Sabio M. Wang X. Marshall P. American Chemical Society: Book of Abstracts. American Chemical Society, Washington, D. C.2004: 22-26Google Scholar). The carboxylate of lumiracoxib forms hydrogen-bonding interactions with Ser-530 and Tyr-385 at the top of the active site. From this crystal structure, it was proposed that the COX-2 selectivity of lumiracoxib arises from the insertion of the methyl group on the phenylacetic acid ring into a small groove provided by the movement of Leu-384 in the COX-2 active site. The movement of this leucine residue is restricted in the COX-1 active site due to the presence of bulky secondary shell residues behind Leu-384 (Ile-525 and Phe-503) that prevent the movement of this residue with inhibitor bound. The corresponding secondary shell residues are Leu-525 in mCOX-2 and Val-525 in hCOX-2 and Leu-503 in both m/hCOX-2 enzymes. The crystal structure failed to elucidate the precise binding sites of the halogens on the lower aniline ring of lumiracoxib, although it appears that either atom could have accessibility to the small hydrophobic pocket utilized by the 2′-methyl group of indomethacin (Ala-527, Val-349, Ser-530, and Leu-531). Although the selectivity of lumiracoxib for COX-2 has been determined in vivo and the crystal structure of lumiracoxib-bound mCOX-2 has been solved, structure-activity relationship studies (SARs) have not been performed, and the chemical and structural basis for the balance that exists between potency and COX-2 selectivity remains unknown for this inhibitor. Therefore, we undertook the synthesis and characterization of a number of lumiracoxib analogs in an attempt to uncover the chemical determinants for selective COX-2 inhibition. In addition, the molecular basis for the selectivity of lumiracoxib was investigated by probing these chemical derivatives against Val-349 and Ser-530 mutants in the COX-2 active site. Materials and Enzymes−The starting materials, ligands, and catalysts for all chemical reactions were commercially available and were purchased from Sigma and Alfa Aesar (Ward Hill, MA). The synthesis and purification of both methylated and des-methyl lumiracoxib analogs was carried out as described in the supplemental materials with only minor modifications to the reaction conditions being necessary for individual compounds. The expression and purification of murine COX-2 and human COX-2 from insect cells and of ovine COX-1 from ram seminal vesicles was performed according to published methods (10.Rowlinson S.W. Crews B.C. Lanzo C.A. Marnett L.J. J. Biol. Chem. 1999; 274: 23305-23310Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Site-directed mutagenesis on murine COX-2 to generate V349A, V349I, V349L, and S530A mutants was performed as described by Prusakiewicz et al. (8.Prusakiewicz J.J. Felts A.S. Mackenzie B.S. Marnett L.J. Biochemistry. 2004; 43: 15439-15445Crossref PubMed Scopus (49) Google Scholar). [1-14C]AA was purchased from PerkinElmer Life Sciences. Synthesis and Structural Characterization of Lumiracoxib Analogs−Various 5′-methylated and des-methyl lumiracoxib analogs were synthesized as described in the supplemental materials (sections A and B) according to supplemental Schemes 1 and 2. Following synthesis, the mass of the purified product was confirmed by electrospray ionization mass spectra, and the structure was confirmed by one-dimensional 1H NMR. The purity of the product was assessed by high performance liquid chromatography (HPLC) using a light scattering detector. The chemical characterization of each lumiracoxib analog is summarized in the supplemental materials (section C). Enzymes−All activity or inhibition studies were performed in 100 mm Tris-HCl buffer containing 500 μm phenol with hematin-reconstituted protein. All inhibitors were dissolved in Me2SO. Reactions were run with hematin-reconstituted proteins at final enzyme concentrations adjusted to give ∼30–35% substrate consumption (hCOX-2 = 145 nm, mCOX-2 = 63 nm, oCOX-1 = 22.5 nm, S530A = 165 nm, V349A = 250 nm, V349I = 268 nm, and V349L = 113 nm). Competitive Inhibition Assays for COX−Several assays were used to determine whether the various lumiracoxib analogs were competitive inhibitors of COX. In one assay, the dual administration of inhibitor and different concentrations of [1-14C]AA (6–50 μm) was performed following a 3-min equilibration of enzyme at 37 °C. In another method, inhibitor was prebound to enzyme for 3 min at 37 °C, followed by the addition of [1-14C]AA at different concentrations for 30 s. In an additional assay that examined the reversibility of COX inhibition, [1-14C]AA was added at a range of concentrations and incubated with inhibitorbound enzyme for different times (30 s to 5 min) to assess the ability of the substrate to compete off the inhibitor. All assays were terminated and analyzed for substrate consumption by TLC as previously described (11.Kalgutkar A.S. Crews B.C. Rowlinson S.W. Marnett A.B. Kozak K.R. Remmel R.P. Marnett L.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 925-930Crossref PubMed Scopus (247) Google Scholar). The values reported were the average of two or more independent determinations. COX Inhibition Screening Assay−Concentration-dependent inhibition reactions were performed by preincubating the inhibitor and enzyme for 17 min at 25 °C, followed by 3 min at 37 °C prior to the addition of 50 μm [1-14C]AA for 30 s at 37 °C. Assays were terminated and analyzed for substrate consumption by TLC as described above. All inhibitor concentrations for 50% enzyme activity (IC50) were determined graphically using Prism and were the average of at least two independent determinations. Time-dependent COX Inhibition Assays−Time-dependent inhibition assays were conducted by preincubating increasing concentrations of the inhibitor with m/hCOX-2, oCOX-1, or the Val-349 mutants (V349A, V349I, and V349I) for various times (0, 0.125, 0.25, 0.5, 1, 3, 5, 15, 30, and 60 min) at 37 °C prior to the addition of 50 μm [1-14C]AA for 30 s at 37 °C. Reactions were terminated and analyzed by TLC as described above. The values of the kinetic parameters were the average of three independent determinations. Synthesis of Lumiracoxib and a Series of Lumiracoxib Analogs−A series of lumiracoxib analogs that varied in the substituents on the phenylacetic acid ring and on the lower aniline ring were synthesized (Table 1). Lumiracoxib itself (compound 1) was synthesized according to a previously published multistep synthetic method that is described in Scheme 1 of the supplemental materials (12.Acemoglu M. Allmendinger T. Calienni J. Cercus J. Loiseleur O. Sedelmeier G.H. Xu D. Tetrahedron. 2004; 60: 11571-11586Crossref Scopus (24) Google Scholar). The various structural analogs of lumiracoxib were also synthesized according to Scheme 1 (methyl derivatives; illustrated for compound 1) or Scheme 2 (des-methyl derivatives; illustrated for compound 11). Mass spectral analysis, one-dimensional proton NMR spectroscopic, and HPLC analyses were performed on each of the lumiracoxib analogs to confirm both the structure and purity of the compounds. The 1H NMR spectrum of each lumiracoxib derivative was compared with previously published spectra where available (12.Acemoglu M. Allmendinger T. Calienni J. Cercus J. Loiseleur O. Sedelmeier G.H. Xu D. Tetrahedron. 2004; 60: 11571-11586Crossref Scopus (24) Google Scholar, 13.Moser P. Sallmann A. Wiesenberg I. J. Med. Chem. 1990; 33: 2358-2368Crossref PubMed Scopus (182) Google Scholar), and each of the analogs was shown by HPLC analysis with evaporative light scattering detection to elute as a single peak. The spectral characterization of each lumiracoxib analog is provided in the supplemental materials.TABLE 1Determination of IC50 values of lumiracoxib analogs with COX Each lumiracoxib analog was screened against purified oCOX-1, mCOX-2, and hCOX-2 as described under “Experimental Procedures” for COX inhibition assays. The lined symbol indicates less than 20% inhibition up to inhibitor concentrations of 4 μm. Where appropriate, the values in parentheses indicate the extent of inhibition (where the plateau for inhibition is reached) associated with each inhibitor. Evaluation of COX Inhibition by Lumiracoxib−The kinetic basis for lumiracoxib inhibition was determined. Following a 3-min equilibration of purified, hematin-reconstituted m/hCOX-2 or oCOX-1 at 37 °C, lumiracoxib and [1-14C]AA were added simultaneously and incubated for 30 s. No significant inhibition was observed over a wide range of inhibitor and substrate concentrations, indicating that lumiracoxib is not a pure competitive inhibitor (supplemental materials, section D, Fig. 1). We next tested lumiracoxib against purified m/hCOX-2 and oCOX-1 using a standard time-dependent protocol designed to determine IC50 values for enzyme inhibition. Fig. 3A shows the inhibition curve for lumiracoxib (compound 1). Lumiracoxib did not inhibit oCOX-1 to any appreciable extent, consistent with prior studies (14.Esser R. Berry C. Du Z. Dawson J. Fox A. Fujimoto R.A. Haston W. Kimble E.F. Koehler J. Peppard J. Quadros E. Quintavalla J. Toscano K. Urban L. van Duzer J. Zhang X. Zhou S. Marshall P.J. Br. J. Pharmacol. 2005; 144: 538-550Crossref PubMed Scopus (105) Google Scholar). Unexpectedly, however, lumiracoxib proved to be a poor inhibitor of both mCOX-2 (∼15% inhibition) and hCOX-2 (∼50% inhibition). Prior in vitro and in vivo experiments with lumiracoxib have demonstrated that the inhibitor exhibits slow, time-dependent inhibition of purified hCOX-2 with a Ki of 60 nm and IC50 values of 130 and 140 nm, respectively, for the inhibition of COX-2 in cell-based assays and human whole blood assays (14.Esser R. Berry C. Du Z. Dawson J. Fox A. Fujimoto R.A. Haston W. Kimble E.F. Koehler J. Peppard J. Quadros E. Quintavalla J. Toscano K. Urban L. van Duzer J. Zhang X. Zhou S. Marshall P.J. Br. J. Pharmacol. 2005; 144: 538-550Crossref PubMed Scopus (105) Google Scholar). In our study, the inhibition of hCOX-2 activity plateaus at ∼50% inhibition and displays characteristics of a reversible inhibitor. Although the extent of inhibition of the human isoform is only 50%, our results do support the fact that lumiracoxib exhibits selectivity for hCOX-2 over COX-1, since no appreciable inhibition of oCOX-1 was observed even with very high concentrations of inhibitor (100 μm). The standard IC50 assay described above uses levels of arachidonic acid (50 μm) that are well above saturation. Thus, if inhibition of m/hCOX-2 by lumiracoxib is readily reversible, lumiracoxib would not be expected to strongly inhibit the COX activity at high substrate concentrations. To probe the effect of substrate concentration on enzyme inhibition, we incubated lumiracoxib with different concentrations of murine or human COX-2 for 3 min at 37 °C prior to the addition of [1-14C]AA (6–50 μm) for 30 s. Fig. 3B shows that lumiracoxib competes moderately with substrate at 50 μm AA but inhibits hCOX-2 to nearly 100% at high inhibitor concentrations and low arachidonate concentrations. Fig. 3C shows a similar profile for lumiracoxib and mCOX-2. Lumiracoxib is a weaker inhibitor of the murine enzyme, as demonstrated by the low extent of inhibition at 25 and 50 μm AA. In addition, lumiracoxib was not able to fully inhibit mCOX-2 even at the lowest substrate concentrations and highest inhibitor concentrations. Lumiracoxib, at a concentration of 1 μm, was prebound to purified enzymes for 3 min at 37 °C, followed by the addition of arachidonate at 50 μm for increasing times (30 s to 5 min). The time course for oxidation of arachidonate is a reflection of the dissociation of lumiracoxib; it exhibited rate constants of 0.0098 s-1 and 0.020 s-1 for hCOX-2 and mCOX-2, respectively (Fig. 4). These results corroborate the inhibition plateaus (15% for mCOX-2 and 50% for hCOX-2) that were initially observed with the IC50 screen and indicate a more rapid reversibility for the murine enzyme. Diclofenac was tested in the same assay and showed off-rates from murine and human COX-2 in the range of 2 × 10-5 s-1, suggesting a much more tightly bound inhibitor. The Kinetics of COX Inhibition by Lumiracoxib−The dependence of COX inhibition by lumiracoxib on time and inhibitor concentration was determined by adding arachidonic acid to wild-type COX preparations following preincubation with inhibitor for various times. The decrease in substrate conversion at different inhibitor concentrations was plotted against the preincubation times and fit to a single-exponential decay with a plateau to determine a value for kobs. The dependence of kobs on inhibitor concentration is represented by Equation 1, where KI corresponds to the inhibitor concentration that yields a rate equal to half the limiting rate, and k2 represents the limiting forward rate constant for inhibition (8.Prusakiewicz J.J. Felts A.S. Mackenzie B.S. Marnett L.J. Biochemistry. 2004; 43: 15439-15445Crossref PubMed Scopus (49) Google Scholar). The reverse rate constant, k-2, is equal to the y intercept and is zero for compounds that display functionally irreversible inhibition. kobs=((k2×[I])/(KI+[I]))+k-2(Eq.1) Table 2 shows the kinetic parameters for the time- and concentration-dependent inhibition of wild-type COX enzymes by lumiracoxib. The values for the inhibition of hCOX-2 were in good agreement with previously reported values of KI (0.06 μm) and k2 (0.005 s-1) (14.Esser R. Berry C. Du Z. Dawson J. Fox A. Fujimoto R.A. Haston W. Kimble E.F. Koehler J. Peppard J. Quadros E. Quintavalla J. Toscano K. Urban L. van Duzer J. Zhang X. Zhou S. Marshall P.J. Br. J. Pharmacol. 2005; 144: 538-550Crossref PubMed Scopus (105) Google Scholar). Since different experimental assays were used to determine the kinetic parameters for COX inhibition, the present values of KI and k2 for lumiracoxib inhibition of hCOX-2 are slightly higher (0.32 μm and 0.087 s-1) than the values noted above. As indicated by a plateau at 50% activity remaining in the IC50 curve, the time-dependent inhibition of hCOX-2 by lumiracoxib resulted in a measurable k-2 (0.0017 s-1), which indicated that the second binding step was reversible. This k-2 is close to the off-rate for lumiracoxib from hCOX-2 measured from the time course of arachidonic acid oxygenation described above (0.0098 s-1) (Fig. 4). Not surprisingly, the kinetic parameters for the inhibition of murine COX-2 by lumiracoxib could not be determined at either room temperature or 37 °C due to poor inhibition of this enzyme by lumiracoxib at high arachidonate concentrations. This is in agreement with the attempted IC50 determination for lumiracoxib inhibition of mCOX-2 where a plateau was reached at 85% remaining activity.TABLE 2Kinetic parameters of time-dependent inhibition of COX enzymes and mutants by lumiracoxib (1.Lyseng-Williamson K.A. Curran M.P. Drugs. 2004; 64: 2237-2246Crossref PubMed Scopus (19) Google Scholar) and compound 2 Kinetic parameters ± S.E. were determined from inhibition assays outlined under “Experimental Procedures” and shown in Equation 1. Increasing concentrations of inhibitors were preincubated with enzyme for various times (0–60 min) at 37 °C prior to the addition of substrate (50 μm). Data points were fit to nonlinear regression analysis, and the kobs values were plotted against inhibitor concentration in a secondary plot to obtain the kinetic parameters shown above. −, a reverse rate constant (k-2) that was effectively zero. ND, a rate constant that could not be determined.K1k2k-2μms-1s-11 (hCOX-2)0.32 ± 0.110.087 ± 0.0070.0017 ± 0.00061 (mCOX-2)NDNDND1 (mV349I)0.75 ± 0.380.107 ± 0.012−2 (hCOX-2)1.2 ± 0.260.074 ± 0.005−2 (mCOX-2)0.82 ± 0.230.069 ± 0.004−2 (mV349I)1.1 ± 0.250.048 ± 0.003−2 (oCOX-1)2.3 ± 0.610.078 ± 0.0050.0025 ± 0.0003 Open table in a new tab SAR Requirements for Lumiracoxib Potency and COX-2 Selectivity−The COX-2 selectivity of lumiracoxib has been attributed to the methyl group on the phenylacetic acid ring of the inhibitor and its insertion into a small pocket around Leu-384, which is made accessible by small secondary shell residues that exist only in COX-2 (9.Clark K. Kulathila R. Koehn J. Rieffel S. Strauss A. Hu S. Kalfoglou M. Szeto D. Lasala D. Sabio M. Wang X. Marshall P. American Chemical Society: Book of Abstracts. American Chemical Society, Washington, D. C.2004: 22-26Google Scholar). The role of the halogen substituents on the lower aniline ring and the nature of their interactions with COX-1 or COX-2 has not been investigated. Hypothetically, the fluorine or chlorine substituents of lumiracoxib may also contribute to COX-2 selectivity and/or to the potency of inhibition. Purified and hematin-reconstituted hCOX-2, mCOX-2, and oCOX-1 were screened against lumiracoxib and the various synthetic analogs (compounds 1–22; Table 1) in our standard time dependence IC50 assay. Several compounds were additionally screened in separate assays to rule out COX inhibition at low arachidonate concentrations (data not shown). Fig. 5 shows the inhibition curves for diclofenac (A), lumiracoxib (B), and three lumiracoxib analogs (C–E) against purified COX-1 and COX-2. As expected from previously reported inhibition studies (7.Rowlinson S.W. Kiefer J.R. Prusakiewicz J.J. Pawlitz J.L. Kozak K.R. Kalgutkar A.S. Stallings W.C. Kurumbail R.G. Marnett L.J. J. Biol. Chem. 2003; 278: 45763-45769Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), diclofenac (compound 10) was a potent, nonselective inhibitor of all three cyclooxygenase isoforms. Replacement of the hydrogen with a methyl group at the 5′-position on the phenylacetic acid ring of diclofenac yields compound 2. Compound 2 appears to reversibly inhibit COX-1 (50% inhibition) but potently inhibits both mouse and human COX-2 in a functionally irreversible manner (nearly 90% inhibition) with IC50 values of 63 and 47 nm, respectively (Fig. 5D and Table 1). From these data, it appears that the addition of the methyl group on the phenyl acetic acid ring of compound 2 introduces COX-2 selectivity. In support of this hypothesis, a des-methyl lumiracoxib analog (compound 11) exhibits nonselective inhibition of all COX isoforms (Fig. 5C) compared with the selective hCOX-2 inhibition demonstrated by compound 1. A fluorine-to-chlorine substitution (compound 1 to compound 2) on the lower aniline ring significantly changes the potency of COX inhibition. Compound 1 inhibits hCOX-2 to only 50%, whereas inhibition of hCOX-2 by compound 2 results in nearly 90% inhibition (Fig. 5, B and D). Although diclofenac and compound 11 are both nonselective inhibitors, diclofenac demonstrates potent inhibition against all COX isoforms (∼90%) compared with its 6-fluoro counterpart (25–40%), supporting a role for the chemical substituents on the lower aniline ring in the potent inhibition of COX (Fig. 5, A and C). An additional substitution at the para position on the aniline ring (compound 6) abrogates the COX-2 selectivity observed with compound 2, suggesting that the precise positioning of substituents on the lower ring is required for selective inhibition of COX-2 (Fig. 5E). Replacement of the fluorine atom on lumiracoxib with hydrogen to give the monochlorinated compound 3 negates any of the hCOX-2 selectivity observed with lumiracoxib and yields a compound that is a very poor inhibitor of all three COX enzymes (Table 1). The monofluorinated compound 4 and the nonhalogenated compound 5 were similarly shown to be very poor, nonselective COX-1/2 inhibitors in this assay. The des-methyl versions of these mono- or nonhalogenated derivatives (compounds 12–14) showed identical results (Table 1), suggesting that a 2,6 substitution on the lower aniline ring is required for COX inhibition. However, derivatives that contained a 2,6-difluoro or 2-fluoro-6-methyl substitution did not inhibit any of the COX isoforms (compounds 7, 8, 15, and 16). Interestingly, a 2-chloro-6-methyl (compounds 9 and 17) or 2,6-dimethyl (compound 20) substitution restored potent and time-dependent COX inhibition. The addition of larger alkyl groups, such as ethyl (compound 21) and isopropyl (compound 22), to the ortho positions on the lower aniline ring resulted in a reduction of inhibitor potency (Table 1). Less potent inhibition of COX also was observed with derivatives that contained a trifluoromethyl substitution. Combined, these data indicate that a 2,6-dichloro, 2,6-dimethyl, or 2-chloro-6-methyl substitution is preferred for potent, time-dependent inhibition of COX. In addition, our SAR analysis corroborates the requirement for a methyl group on the phenylacetic acid ring to generate COX-2 selectivity when there is an appropriate 2,6 substitution on the lower aniline ring. Interestingly, a comparison between the 2,6-difluoro (compound 7) and 2-fluoro-6-chloro and (compound 1) substitution suggests a contributory role for the chlorine atom in COX-2 selectivity. Compound 7 does not inhibit COX, whereas lumiracoxib (compound 1) is selective for hCOX-2 over oCOX-1. This potential role for the chlorine atom in selectivity is further supported by a comparison between compounds 8 and 9 (a 2-fluoro-6-methyl substitution versus a 2-chloro-6-methyl substitution). Here again, the replacement of a fluorine atom with a chlorine" @default.
• W2000748742 created "2016-06-24" @default.
• W2000748742 creator A5072181271 @default.
• W2000748742 creator A5089031929 @default.
• W2000748742 date "2007-06-01" @default.
• W2000748742 modified "2023-10-14" @default.
• W2000748742 title "Molecular Determinants for the Selective Inhibition of Cyclooxygenase-2 by Lumiracoxib" @default.
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