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  MolecularDeterminantsfortheSelectiveInhibitionof Cyclooxygenase-2byLumiracoxib * □ S Receivedforpublication,October20,2006,andinrevisedform,April12,2007  Published,JBCPapersinPress,April12,2007,DOI10.1074/jbc.M609883200 AnnaL.BlobaumandLawrenceJ.Marnett 1 FromtheA.B.HancockJr.MemorialLaboratoryforCancerResearch,DepartmentsofBiochemistry,Chemistry,andPharmacology,VanderbiltInstituteforChemicalBiology,CenterinMolecularToxicology,andVanderbilt-IngramCancerCenter,VanderbiltUniversitySchoolofMedicine,Nashville, Tennessee 37232 LumiracoxibisthefirstexampleofamarketedCOX-2inhibitorofthearylaceticacidclass,anditisreportedtobethemostselectiveCOXIB in vivo .However,themolecularbasisofitsCOX-2inhibi-tionhasnotbeencompletelydefined.Usingstandardassays,lumi-racoxib was found to be a poor inhibitor of purified ovine COX-1and a relatively weak inhibitor of purified human COX-2. TheextentofCOX-2inhibitionplateauedataround50%andsuggestedthat the inhibitor may be reversibly bound to the enzyme. Kineticstudieswithlumiracoxibdemonstratedthatitwasatime-depend-entandslowlyreversibleinhibitorofhumanCOX-2thatexhibitedatleasttwobindingstepsduringinhibition.Derivativesoflumira-coxib were synthesized with or without the methyl group on thephenylaceticacidringandwithvarioussubstitutionsontheloweraniline ring. Inhibition studies demonstrated that the methylgrouponthephenylaceticacidringisrequiredforCOX-2selectiv-ity. The chemical identity and position of the substituents on theloweranilineringwereimportantindeterminingthepotencyandextentofCOXinhibitionaswellasCOX-2selectivity.Mutationof Ser-530toAlaorVal-349toAlaorLeuabolishedthepotentinhi-bitionobservedwithwild-typehumanCOX-2andkeylumiracoxibanalogs. Interestingly, a Val-349 to Ile mutant was inhibited withequalpotencytohumanCOX-2with2,6-dichloro-,2,6-dimethyl-,or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Takentogether with a recent crystal structure of a lumiracoxib-COX-2complex,thekineticanalysespresentedhereinoftheinhibitionof mutantCOX-2sbylumiracoxiballowsthedefinitionofthemolec-ularbasisofCOX-2inhibition. The cyclooxygenase (COX) 2 enzymes COX-1 and COX-2share high sequence homology (60%), have very similar three-dimensional structures, and catalyze the conversion of arachi-donic acid (AA) to prostaglandin H 2 . Lumiracoxib is a highly selective COX-2 inhibitor that is weakly acidic and displays aunique pharmacological profile that includes rapid absorbanceand a relatively short plasma half-life (1). Lumiracoxib displaysa500-foldgreaterselectivityforCOX-2thanCOX-1 invivo (2)and in clinical studies has shown a 3–4-fold reduction in ulcercomplications versus classicalNSAIDs(3,4).Lumiracoxiblacksthe tricyclic structure of the diarylheterocycle class of COX-2-selective inhibitors ( e.g.  celecoxib and rofecoxib) and does notcontainasulfonamideorsulfonegroup.Instead,lumiracoxibisa close structural analog of diclofenac (Fig. 1). Although lumi-racoxibanddiclofenacsharestructuralsimilarities,theyexhibitlarge differences in the selectivity of COX-2 inhibition. Themolecular basis for this difference in the selectivity of COXinhibition is not entirely understood.Previous crystal structures of COX enzymes with carboxylicacid-containing NSAIDs reveal that the inhibitors are typically positioned with their carboxylates coordinated to Arg-120 andtheir aromatic functional groups projecting up into thecyclooxygenaseactivesite(5,6).However,acrystalstructureof diclofenac bound in the active site of COX-2 (Fig. 2  B ) revealedan inverted binding mode of the molecule with its carboxylicacidmoietycoordinatedtoSer-530andTyr-385(7).Structure-activity studies have found that another distinctive bindingmodeisexhibitedbyindomethacin,anonselectiveNSAIDthatis a time-dependent, functionally irreversible inhibitor of COXenzymes (8). Indomethacin binds in the cyclooxygenase activesite with its carboxylic acid group ion-pairing and hydrogen-boding with Arg-120 and Tyr-355 (Fig. 2  A ). In addition, its2  -methyl group binds in a hydrophobic pocket composed of Ala-527, Val-349, Ser-530, and Leu-531 (6). Mutations of Val-349inthismethyl-bindingpockettoalanineorleucinealterthesize of the pocket and lead to an increase or decrease in thepotency of indomethacin, respectively. The 2  - des -methyl ana-logofindomethacindoesnotinhibitCOX-1andisaveryweak,rapidly reversible inhibitor of COX-2 (8).Recently, the crystal structure of murine COX-2 with lumi-racoxib was solved and revealed that lumiracoxib binds in aninverted orientation similar to that of diclofenac (9). The car-boxylate of lumiracoxib forms hydrogen-bonding interactionswithSer-530andTyr-385atthetopoftheactivesite.Fromthiscrystal structure, it was proposed that the COX-2 selectivity of lumiracoxib arises from the insertion of the methyl group onthe phenylacetic acid ring into a small groove provided by themovementofLeu-384intheCOX-2activesite.Themovement *  This research was supported by National Institutes of Health GrantsCA89450, CA119629, and CA09582. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “ advertisement  ” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact. □ S  Theon-lineversionofthisarticle(availableathttp://www.jbc.org)containssupplemental Schemes 1 and 2 and Figs. 1 and 2. 1  To whom correspondence should be addressed: Dept. of Biochemistry,Vanderbilt University School of Medicine, 23rd Ave. South at Pierce,Nashville, TN 37232. Tel.: 615-343-7329; Fax: 615-343-7534; E-mail:larry.marnett@vanderbilt.edu. 2  The 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-inflamma-tory drug; SAR, structure activity relationship; HPLC, high pressure liq-uid chromatography.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 22, pp. 16379–16390, June 1, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. JUNE 1, 2007ã VOLUME 282ãNUMBER 22  JOURNAL OF BIOLOGICAL CHEMISTRY   16379   b  y  on J  ul   y  3  ,2  0  0  8 www. j   b  c . or  gD  ownl   o a d  e d f  r  om  http://www.jbc.org/cgi/content/full/M609883200/DC1Supplemental Material can be found at:  of this leucine residue is restricted in the COX-1 active site dueto the presence of bulky secondary shell residues behind Leu-384 (Ile-525 and Phe-503) that prevent the movement of thisresidue with inhibitor bound. The corresponding secondary shell residues are Leu-525 in mCOX-2 and Val-525 in hCOX-2andLeu-503inbothm/hCOX-2enzymes.Thecrystalstructurefailed to elucidate the precise binding sites of the halogens onthe lower aniline ring of lumiracoxib, although it appears thateither atom could have accessibility to the small hydrophobicpocket 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 beendetermined  in vivo  and the crystal structure of lumiracoxib-bound mCOX-2 has been solved, structure-activity relation-shipstudies(SARs)havenotbeenperformed,andthechemicalandstructuralbasisforthebalancethatexistsbetweenpotency 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 thechemicaldeterminantsforselectiveCOX-2inhibition.Inaddi-tion, the molecular basis for the selectivity of lumiracoxib wasinvestigated by probing these chemical derivatives against Val-349 and Ser-530 mutants in the COX-2 active site. EXPERIMENTALPROCEDURES  MaterialsandEnzymes —Thestartingmaterials,ligands,andcatalystsforallchemicalreactionswerecommerciallyavailableand were purchased from Sigma and Alfa Aesar (Ward Hill,MA). The synthesis and purification of both methylated and des -methyllumiracoxibanalogswascarriedoutasdescribedinthe supplemental materials with only minor modifications tothe reaction conditions being necessary for individual com-pounds.TheexpressionandpurificationofmurineCOX-2andhuman COX-2 from insect cells and of ovine COX-1 from ramseminal vesicles was performed according to published meth-ods (10). Site-directed mutagenesis on murine COX-2 to gen-erate V349A, V349I, V349L, and S530A mutants was per-formed as described by Prusakiewicz  et al.  (8). [1- 14 C]AA waspurchased from PerkinElmer Life Sciences. Synthesis and Structural Characterization of Lumiracoxib Analogs —Various 5  -methylated and  des -methyl lumiracoxibanalogs were synthesized as described in the supplementalmaterials (sections A and B) according to supplementalSchemes 1 and 2. Following synthesis, the mass of the purifiedproductwasconfirmedbyelectrosprayionizationmassspectra,andthestructurewasconfirmedbyone-dimensional 1 HNMR.The purity of the product was assessed by high performanceliquid chromatography (HPLC) using a light scattering detec-tor. The chemical characterization of each lumiracoxib analogis summarized in the supplemental materials (section C).  Enzymes —All activity or inhibition studies were performedin 100 m M  Tris-HCl buffer containing 500   M  phenol withhematin-reconstituted protein. All inhibitors were dissolved inMe 2 SO. Reactions were run with hematin-reconstituted pro-teinsatfinalenzymeconcentrationsadjustedtogive  30–35%substrateconsumption(hCOX-2  145n M ,mCOX-2  63n M ,oCOX-1    22.5 n M , S530A    165 n M , V349A    250 n M ,V349I  268 n M , and V349L  113 n M ). Competitive Inhibition Assays for COX  —Several assays wereused to determine whether the various lumiracoxib analogswere competitive inhibitors of COX. In one assay, the dual admin-istration of inhibitor and differentconcentrations of [1- 14 C]AA (6–50  M ) was performed following a3-min equilibration of enzyme at37 °C. In another method, inhibitorwas prebound to enzyme for 3 minat 37 °C, followed by the addition of [1- 14 C]AA at different concentra-tions for 30 s. In an additional assay that examined the reversibility of COX inhibition, [1- 14 C]AA wasadded at a range of concentrationsand incubated with inhibitor-bound enzyme for different times(30 s to 5 min) to assess the ability of the substrate to compete off theinhibitor. All assays were termi- FIGURE 1.  Structures of the nonselective NSAIDs, diclofenac and indo-methacin, compared with the COX-2-selective inhibitors, lumiracoxib,celecoxib,androfecoxib. FIGURE 2.  Different inhibitory binding modes of two nonselective NSAIDs.  A  shows indomethacin in theactive site of mCOX-2 (6). The  para -chlorobenzyl group is positioned near Tyr-385, and the carboxylate ishydrogen-bonded to Arg-120 and Tyr-355 at the constriction site. The 2  -methyl group of the inhibitor isbound in a small hydrophobic pocket provided by Val-349, Leu-531, Ala-527, and Ser-530. B  shows diclofenacintheactivesiteofmCOX-2(7).Theinhibitorbindsinaninvertedorientationwithitscarboxylateatthetopof the active site making hydrogen bonds with Tyr-385 and Ser-530. Diclofenac does not contact Arg-120 and Tyr-355 at the constriction site. DeterminantsofCyclooxygenase-2InhibitionbyLumiracoxib 16380  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 282ãNUMBER 22ã JUNE 1, 2007   b  y  on J  ul   y  3  ,2  0  0  8 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   nated and analyzed for substrate consumption by TLC aspreviously described (11). The values reported were theaverage of two or more independent determinations. COX Inhibition Screening Assay —Concentration-dependentinhibition reactions were performed by preincubating theinhibitor and enzyme for 17 min at 25 °C, followed by 3 min at37 °Cpriortotheadditionof50  M [1- 14 C]AAfor30sat37 °C.Assays were terminated and analyzed for substrate consump-tionbyTLCasdescribedabove.Allinhibitorconcentrationsfor50% enzyme activity (IC 50 ) were determined graphically usingPrism and were the average of at least two independentdeterminations. Time-dependent COX Inhibition Assays —Time-dependentinhibition assays were conducted by preincubating increasingconcentrations of the inhibitor with m/hCOX-2, oCOX-1, orthe Val-349 mutants (V349A, V349I, and V349I) for varioustimes(0,0.125,0.25,0.5,1,3,5,15,30,and60min)at37 °Cpriorto the addition of 50  M  [1- 14 C]AA for 30 s at 37 °C. Reactionswere terminated and analyzed by TLC as described above. The valuesofthekineticparametersweretheaverageofthreeinde-pendent determinations. RESULTS Synthesis of Lumiracoxib and a Series of Lumiracoxib Analogs —Aseriesoflumiracoxibanalogsthatvariedinthesub-stituents on the phenylacetic acid ring and on the lower anilinering were synthesized (Table 1). Lumiracoxib itself (compound1) was synthesized according to a previously published multi-stepsyntheticmethodthatisdescribedinScheme1ofthesup-plemental materials (12). 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). Massspectral analysis, one-dimensional proton NMR spectroscopic, TABLE1 DeterminationofIC 50 valuesoflumiracoxibanalogswithCOX EachlumiracoxibanalogwasscreenedagainstpurifiedoCOX-1,mCOX-2,andhCOX-2asdescribedunder“ExperimentalProcedures”forCOXinhibitionassays.Thelinedsymbol indicates less than 20% inhibition up to inhibitor concentrations of 4  M . Where appropriate, the values in parentheses indicate the extent of inhibition (where theplateau for inhibition is reached) associated with each inhibitor. DeterminantsofCyclooxygenase-2InhibitionbyLumiracoxib JUNE 1, 2007ã VOLUME 282ãNUMBER 22  JOURNAL OF BIOLOGICAL CHEMISTRY   16381   b  y  on J  ul   y  3  ,2  0  0  8 www. j   b  c . or  gD  ownl   o a d  e d f  r  om   andHPLCanalyseswereperformedoneachofthelumiracoxibanalogs to confirm both the structure and purity of the com-pounds. The  1 H NMR spectrum of each lumiracoxib derivativewas compared with previously published spectra where avail-able(12,13),andeachoftheanalogswasshownbyHPLCanal- ysis with evaporative light scattering detection to elute as asingle peak. The spectral characterization of each lumiracoxibanalog is provided in the supplemental materials.  Evaluation of COX Inhibition by Lumiracoxib —Thekinetic basis for lumiracoxib inhibition was determined. Fol-lowing a 3-min equilibration of purified, hematin-reconsti-tuted m/hCOX-2 or oCOX-1 at 37 °C, lumiracoxib and[1- 14 C]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 lumira-coxib is not a pure competitive inhibitor (supplemental mate-rials, section D, Fig. 1).We next tested lumiracoxib against purified m/hCOX-2 andoCOX-1usingastandardtime-dependentprotocoldesignedtodetermine IC 50  values for enzyme inhibition. Fig. 3  A  shows theinhibition curve for lumiracoxib (compound 1). Lumiracoxibdid not inhibit oCOX-1 to any appreciable extent, consistentwith prior studies (14). Unexpectedly, however, lumiracoxibproved to be a poor inhibitor of both mCOX-2 (  15% inhibi-tion) and hCOX-2 (  50% inhibition). Prior  in vitro  and  in vivo experiments with lumiracoxib have demonstrated that theinhibitor exhibits slow, time-dependent inhibition of purifiedhCOX-2witha  K  i of60n M andIC 50  valuesof130and140n M ,respectively, for the inhibition of COX-2 in cell-based assaysand human whole blood assays (14). In our study, the inhi-bition of hCOX-2 activity plateaus at   50% inhibition anddisplays characteristics of a reversible inhibitor. Althoughthe extent of inhibition of the human isoform is only 50%,our results do support the fact that lumiracoxib exhibitsselectivity for hCOX-2 over COX-1, since no appreciableinhibition of oCOX-1 was observed even with very high con-centrations of inhibitor (100   M ).The standard IC 50  assay described above uses levels of arachidonicacid(50  M )thatarewellabovesaturation.Thus,if inhibition of m/hCOX-2 by lumiracoxib is readily reversible,lumiracoxibwouldnotbeexpectedtostronglyinhibittheCOXactivity at high substrate concentrations. To probe the effect of substrate concentration on enzyme inhibition, we incubatedlumiracoxib with different concentrations of murine or humanCOX-2 for 3 min at 37 °C prior to the addition of [1- 14 C]AA(6–50   M ) for 30 s. Fig. 3  B  shows that lumiracoxib competesmoderatelywithsubstrateat50  M AAbutinhibitshCOX-2tonearly100%athighinhibitorconcentrationsandlowarachido-nate concentrations. Fig. 3 C   shows a similar profile for lumira-coxib and mCOX-2. Lumiracoxib is a weaker inhibitor of themurine enzyme, as demonstrated by the low extent of inhibi-tion at 25 and 50  M  AA. In addition, lumiracoxib was not ableto fully inhibit mCOX-2 even at the lowest substrate concen-trations and highest inhibitor concentrations.Lumiracoxib, at a concentration of 1   M , was prebound topurifiedenzymesfor3minat37 °C,followedbytheadditionof arachidonate at 50  M  for increasing times (30 s to 5 min). Thetime course for oxidation of arachidonate is a reflection of thedissociation of lumiracoxib; it exhibited rate constants of 0.0098 s  1 and 0.020 s  1 for hCOX-2 and mCOX-2, respec-tively (Fig. 4). These results corroborate the inhibition plateaus(15% for mCOX-2 and 50% for hCOX-2) that were initially observedwiththeIC 50 screenandindicateamorerapidrevers-ibilityforthemurineenzyme.Diclofenacwastestedinthesameassay and showed off-rates from murine and human COX-2 inthe range of 2    10  5 s  1 , suggesting a much more tightly bound inhibitor. The Kinetics of COX Inhibition by Lumiracoxib —Thedependence of COX inhibition by lumiracoxib on time andinhibitor concentration was determined by adding arachidonicacid to wild-type COX preparations following preincubationwithinhibitorforvarioustimes.Thedecreaseinsubstratecon- version at different inhibitor concentrations was plottedagainst the preincubation times and fit to a single-exponentialdecay with a plateau to determine a value for  k  obs . The depend-ence of   k  obs  on inhibitor concentration is represented by Equa-tion1,where  K   I  correspondstotheinhibitorconcentrationthat yieldsarateequaltohalfthelimitingrate,and k  2 representsthelimiting forward rate constant for inhibition (8). The reverse FIGURE3. Cyclooxygenaseenzymeinhibitionbylumiracoxib.  A showstheresultsofastandardtime-dependentIC 50 screenforlumiracoxibagainstwild-type( wt  )hCOX-2,mCOX-2,andoCOX-1.Lumiracoxibwaspreincubatedwithenzymefor20minpriortotheadditionofsubstrate(50  M )for30sat37 °Casdescribed under “Experimental Procedures.”  B  and  C   show the results of acompetition assay in which purified protein ( B , hCOX-2;  C  , mCOX-2) was pre-bound with lumiracoxib for 3 min at 37 °C prior to the addition of substrate(6–50  M ) for 30 s. For all assays, reactions were terminated and analyzed by TLC as described under “Experimental Procedures.” DeterminantsofCyclooxygenase-2InhibitionbyLumiracoxib 16382  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 282ãNUMBER 22ã JUNE 1, 2007   b  y  on J  ul   y  3  ,2  0  0  8 www. j   b  c . or  gD  ownl   o a d  e d f  r  om 

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