Indian Journal of Biochemistry & Biophysics Vol. 38, February & April 2001, pp. 56-63

Recognition of cyclooxygenase-2 (COX-2) active site by NSAIDs: A computer modelling study

V Kothekar*, S Sahi, M Srinivasan, A Mohan and J Mi shra Department of Biophysics, All India Institute of Med ical Sciences, Ansari Nagar, New Delhi 11 0029, India

Accepted J October 2000

The energetics and models of COX-2 complexed with nonstero idal an ti -innammatory drugs (NSAlDs) ha'ling different degrees of selectivity for two isoforms of COX (COX-2 and COX-I) have been studied using computer modelling ap­proach. The models are obtained for complexes of NS398 (NS), a selective COX-2 inhibitor; indoprofen (lnd), a non­selective inhibitor; di-tert-butylbenzofurans (DHDMBFs) with substituents at the 5th position: CONH(CHc) ~OMe (BFl) , CONH-c-Pr (BF2), 3-methylene-y- butyrolactonyl (BF3) and oxicams namely, mel ox icam (Mel), piroxicam (Pir) and tenoxicam (Ten). These were optim ized using molecular mechanics (MM) and molec ular dynami cs (MD) tec hn iques. The binding energies and struct ures were compared with pharmaco logical parameters and available results with COX-I . In case of NS a larger difference in the binding energies between COX-2 and COX-I wa noticed as compared to that of Ind. It also had stronger interaction with Hi s90 and Tyr355 which is considered important fo r COX-2 selecti vity. There was a difference in the compactness at the channel ent rance between COX-2 selec ti ve and non-se lective li ga nds. Models with DHDMBFs and ox ic:.tms showed a similar corrcl ation. Thc results were used to design a peptide inhibitor, Tyr-Arg-Cys-A l a-~Phe-Cys

(Pcpt ) whi ch could fit beller in the COX-2 cav ity. As per our MD simulation results thi s peptide inhibitor showed both highcr activity and COX-2 select ivi ly.

Introduction Cyclooxygenase (COX) catalyses the first

committed step in arachidonic cascade i.e., the conversion of arachidonic acid to prostaglandin G2 (PGG2) via capture of molecular oxygen and its subsequent conversion to prostaglandin H2 (PGH2) in heme dependent peroxide reduction 1-4. Bei ng natural targets for NSAIDs these are important clinically. Two isoforms of COX (COX-l and COX-2) are recognized. COX-I is present in almost all the ti ssues of the body and its inhibition leads to gastrointes tinal (Gl) disorders, kidney and li ver failure2

•5

.6

. COX-2 occurs in limited number of ti ssues. Its sy nthesis is regulated by specific stimuli7-') which are also responsibl e for elevated levels of PG' s during inflammation 10.1 I. A selecti ve inhi bitor of COX-2 would avoid undesirable side effects during prolonged NSAID's administration. Few compounds have been identified which show COX-2 selectivity I 1-13. However, usually they have low potency. Designing a selective inhibitor for COX-2 is not an easy task, as both the active site dimensions and amino acid side chains in the active cavity do not show much

"'Aulhor for correspondence. E:lllail: kmhdar a hOlmail.l·olll, k ll lh c br@ lll cclin ~ t. e rnet.in; Voi ce phone: 91-011-6593215; Fax : 91-0 I 1-6862663

differences I4-t8. Only, the COX-2 ac tive site is slightly

roomy with a small bulge above Arg513 and His90. Genetic modification data I9

-24

, as also kinetic d 25-30 . I f I d .. ata . suggest a major ro e 0 struct Ta ynamlcs 111

COX-2 selectivity. Interaction of Ty r355 with the ligands, as also the compactness at the channel entrance resulting from the H-bonding network amongst Arg 120, Tyr355, Glu524 and Arg513 were

. d d bid . f' . f' . ~9)0 const ere to e t le eterm tnan ts 0 speci ICIty-" . The membrane-binding loop (residues II I -1 22) and residues 353-356 showed some differences in X-ray data of different ligands with COX-2 and COX-114.17.18. However, these were too small , poss ibly because of the low reso lution (2.9 to 3.3 A) of these data to explain the observed kinetic differences. Hence, in the present paper, we have attempted to exami ne these differences using computer modelling techniques, inclusive of MM and MD. Our aim is to understand energetics and structurJ I differences in different models of COX-2 complexed with COX-2 selective and non-selective ligands.

For this purpose we selected NS, a COX-2 selective ligand 31 ; Ind, a non-selective COX­inhibitol)2; DHDMBFs (EFI, EF2, BF3) dual COX and 5-LOX (5-lipoxygenase) inhibitors3J and oxicams (Mel, Pir and Ten) which showed eli fferent degree of COX-2 selectivit/4

• EFI produced greater reduction

KOTHEKAR el 01. : RECOGNITION OF CYCLOOXYGENASE-2 (COX-2) ACTIVE SITE 57

in swelling followed by BF3 and BF2. BFl also has the greatest selectivity for COX-2 but BF3 is more potent (Table I) . Oxicams are potent, long acting NSAlDs belonging to the class of enolic acid with plasma half-life 50-70 hr, permitting a single daily administration. Mel has greater ill vitro and in vivo inhibitory action against COX-2. The animal model s and comparative clinical trials showed it to be equally potent as diclofenac and naproxen. Pir is a non­selective reversible COX inhibitor which also decreases rheumatoid factor and is hence, useful in rheumatoid arthritis. Ten is quite similar to Pir. The chemical structures of these ligands are depicted in Fig. I .

The correlation between energetics, structures and points of contact with pharmacological activity of these molecules is given. The comparison of these parameters with the results on COX-I obtained by us earlier35

.36 is also discussed in this paper. The results , thus obtained are used to design a peptide inhibitor: Tyr-Arg-Cys-Ala-~Phe-Cys (Pept) belonging to

XC(Z)PC core structure. Our objective is to design a molecule which would occupy a larger volume of the active cavity, has a stronger interaction with the enzyme and also makes a number of specific contacts with the amino acid side chains in the COX-2 cavity. Similar peptides have been found to be involved in anti-inflammatory processes37

. These inhibit the binding of a4~ I (integrin adhesion receptor) to VCAM-J (Vascular Cell Adhesion Molecule-I) at sub­molar concentration38

. The peptide inhibitor (Pept) was cyclized through a disulfide linkage between Cys3-Cys6 seen in Circular Dichroism and Nuclear Magnetic Resonance spectroscopic data on a similar peptide37

namely, (LRCDPC). We incorporated amino acid a-~ dehydro Phe (~Phe) with double bond between Ca-C~ atoms to increase the stability. This would also help in prevention of rapid degradation of the peptide by non­speci fic peptidases. Conformational characteristics of such peptides had been extensively studied by Singh el

ae9. These were considered to be suitable for designing

COX-2 inhibitors40.

Table I-£:omparison of interacti on energy (kCal!mole) of different li gands with volume and pharmacological activity (IC50

COX-2 and rc50 COX-II COX-2)

[The energy values without the bracket are average values during MD study while those inside the bracket are obtained in EM study. ICso for NS and Ind are from references 31 and 32, for 8FI-BF3 from 33 and oxicams from 34]

Ligand IC50 COX-2 IC50 COX-II Vol A3 COX-2 His90 Argl20 Tyr355 Tyr385 COX-2

NS 1.77 42 11 87 -53.8 -5.8 -2.5 -4.7 0.0 (-48.3) (-7.7) (0.5 1 ) (-4.9) (0.0)

Ind 0.5 0.8 1220 -49.4 -0.5 -2.6 -0.9 -1.7 (57.1 ) (-0.6) (-6.3) (-2.1) (-3.4)

BFl 0.03 33 1013 -43.8 -4.6 -4.65 -3.2 -0.08 (-38.5) (-1.19) (-3.5) (-4.18) (-0.14)

8F2 0.55 0.53 1211 -37.0 -1.66 -4.84 - 2.23 -0.14 (-37.0) (-1.91) (-6.68) (-2.02) (-0.02)

8F3 0.003 12.86 1260 -39.4 -2.16 -6. 11 -2.99 -0. 12 (-35.3) (-2.42) (-5.2) (-3.0 1) (-0.00)

Mel 0.17 1.25 1225 -54.2 -0.39 -0.76 -4.28 - 1.46 (-54. 1) (-1.6) (-0.31 ) (-1.94) (-4.14)

Pir 0.60 0.004 1182 ( -6 1.5) (-0.27) (-1.37) (-3.09) (-2.03)

Ten 1132 (- 58.2) (-0.12) (-0.965) (-3.27) (-1.28)

Pept 2090 -89.96 -4.89 -11.17 -6.94 -0.461 (- 122) (-4.06) (-3.75) (-8.67) (-2.95)

58 IND IAN J. 1310CHEM. BIOPHYS., VOL. 38, FEBRUARY & APRIL 2001

Fig. I- Nome nclature :li1d ro tati onal angles in NS, Ind , Pept, 13 F t, 13F2, lIF3, Mel, Pir and Ten . The hydroge n atoms attached to carbon are not shown. Carbon alo m numbering for lIF2 and Pir is same as for lIFl and Mel.

We pre-screened conformational flexibility of all the li gands (li sted in Table I) by systematic search using simulated annealing molecu lar dynamics (SAMD) and MD methods in vacuum and in aqueous environment. Minimum energy conformers were docked in the active cavity of COX-2. The complex structures were optimized by MM and MD simulations for 250 pico seconds (ps) using AMBER 5.0. The structural data from selected files as also energy partitioning was analyzed and compared with the pharmacological parameters listed in Table I.

Methodology

X-ray data on COX-2 (native, as well as with flurbiprofen) was used for obtaining the starting models . These structures (I PRH and 4 COX) were downloaded from protein data bank41. The ligands were docked in the acti ve cavity using graphic tools in MOLMOL 42. Complex geometri es were refined using our energy grid program IMFl 43

. The flex ible torsional angles in the ligands (shown in Fig.l) and in amino acid side chains of the enzyme in its active cavity were allowed to rotate. The detailed docking procedure used in each case, has been di scussed in our earlier papersJ5.36

.44. Both MM and MD simulations were carried out using Sander's module of AMBER 5.0-15. We used all atom force field for the

li gands and united atom force field for the enzyme. The force field parameters were taken most ly from PARM 96. DAT of AMBER 5.0. Atomic charges were calculated by MOPAC7.046

. The free li gands were dipped in the Monte Carlo equil ibrated water bath with TIP3P water model-17. Distanc dependent di­electric constant with scali ng factor for 1-4 electrostatic interaction equal to two was used for all the complexes. These were energy mi nimi zed (l00 steps of steepest descent follo wed by conjugate energy minimization) in 15000 cycles. MD simulations were carri ed out using non-bonded cut-off distance as 8.0 A. Non-bonded pair-li sts were upgraded after every 20 cycles. Integration was carried out using, Verlet' s algori thm and Leap Frog approx imat ion with time step of 0.001 ps. Complexes were slowly heated over 30 ps to 300 K. These were eq uilibrated for 11 0 ps and simu lated for 11 0 ps after equilibrat ion. The total simul ation time was 250 ps. Potenti::t1 energy initially (during h ming) showed a rise. Later, it dropped by 5-10 % in the first 60 ps of MD simulation. After 120- 140 ps, energy was ve ry stable with mean root mean square deviation (RMSD) of 20-24 kCalimole (less than 0.5%). We selected a 110 ps region (from 140 to 250 s) for detailed analysis. A total number of 55 sub-averaged files at 2 ps time interval were pulled out for ca lcul atio n of structure based parameters. Graphic display was done using MOLMOL 42 and RASMOL -18.

Results and lDiscussion

The flexibility of the li gands was substantia lly reduced in the complexes. The peptide was fai rly stable except for N-terminal Tyr-Arg group.

The active cavities of COX-I and COX-2 extend from the membrane binding region (loop of residues 111 - 120), th rough a narrow entrance restricted by H­bonding network between side chains of Arg 120, Glu524, Tyr355 and Arg5 13 (only in case of COX-2), to Tyr385 at the apex of the channel. The heme group is located above Tyr385. Most of the li gands spanned between Tyr355 to Tyr385 (Fig. 2a,b). These made several specific contacts depending on their dimensions and chemical nature of the functional groups. In COX-l , the channel can be divided into two regions as: region-I , extendi ng from a hydrophobic pocket around Tyr385 ending at Glu524 and region 2, extending from Ser530 to Arg120 and a little portion below it. In COX-2, because of few amino ac ids changes (Ile523-Val523 and Hi s5 13 -Arg513) the channel forks from the membrane end

KOTHEKAR el 01. : RECOGNITION OF CYCLOOXYGENASE-2 (COX-2) ACTIVE SITE 59

(a) (b)

aem

Phe381

Ser530

Phe518

Arg120 vaJ Val116

Tyr355

Fig. 2--(a): A snapshot of Mel with COX-2 at 180 ps of MD simulation. Important res idues are shown in darker lines. (b): Same as Fig. 2a. Shown here are only neighboring residues contacting Mel. The drug (Mel) and Heme group are shown by thicker lines. so al so important side chains Tyr385. Arg 120, Tyr355. Arg513 and Hi s90.

creating an extra space above His90 and Arg513 (region 3). This is supposed to be occupied by COX-2 selective ligands . We monitored the energetics of interaction of ligands with COX-2 using RUNANAL module of AMBER 5.0, using both energy minimised (EM) co-ordinates and MD trajectories during 140-250 ps. Interaction energies of the ligands with COX-2 (whole enzyme) and few important side chains (Hi s90, Arg 120, Tyr355 and Tyr385) in EM model and average during MD are reported here (Table I) along with the inhibitory concentration at 50 % (ICso) values for COX-2 and ratios of ICso COX-II ICso COX-2. These correspond to COX-2 activity and selectivity respectively. In the same table we give mean volume calculated on the basis of number of water molecules expelled from TIP3P Monte Carlo equilibrated water bath .

We notice some differences in the binding energies of the li gands with COX-2 during EM and MD. Thus for examp le the lower va lues are observed in MD in case of NS, BFl and BF3; of the same order in BF2 and Mel and higher in case of Ind and Pept. (MD results for Pir and Ten are not yet available). The

oxicams (Mel, Pir, Ten) had lower binding energies (-54.2,61.5 and -58 .2 kCal/mole) as compared to NS (-53.8 kCal/mole), Ind (-49.4 kCal/mole) and DHDMBFs (-37 to -43.8 kCallmole) . The main reason for this difference in the binding energies of various ligands to COX-2 was the variation in the lengths of the ligands which were: oxicams (14.2-14.8 A); Ind (12.7 A), DHDMBFs (9-10 A) and NS (about 8 A). Other important factors were their dimensions which were perpendicular to the length of the channel. This led to lower interaction energy for NS as compared to Ind.

Comparison of ICso values with the interact ion energies of the ligands is rather disappointing. In the case of the ligands with different chemical characteristics, there seems to be no correlation what so ever between the two. Thus NS is far less potent than Ind, but had lower binding energy. DHDMBFs are far more potent than oxicams but have higher interaction energy. This contradiction is not unexpected as ICso values are highly dependen t on the avai labililty of the ligand for the interaction with the enzyme, experimental model s used and conditions of

60 INDIAN J. BIOCHEM. BIOPHYS .. VOL. 38, FEBRUARY & APRIL 2001

the experiment. Within the same series of drugs, when same experimental models were used for the calculation of ICso values, some correlation was observed viz BFI is more potent than BF2 and also has lower interaction energy. But this is not the rule. Mel has higher binding energy than Pir but is more potent. The discrepancy is partly because of experimental errors in both the techniques. However, the main reason is neglect of entropic contributions in comparison of the binding energies. The mean values obtained by MD method are no doubt better. Correlation of ICso values with volumes seems to be better than binding energies. The changes are in the

, right direction, in case of NS, BF2, Ind, Pir and Mel. The enzyme selectivity can be monitored, by comparing the ICso values of the two isoforms with relative binding energies. Only available results for COX-l binding are with NS and Ind35

.36. These showed that the interaction energies are respectively -42.6 and -50 kCallmole. A larger difference in the interaction energies is observed for NS, a COX-2 selective inhibitor.

The most important factor governing both potency as well as enzyme selectivity of NSAIDs, is the interaction between the amino acid side chains of the enzymes in the active cavity and functional groups in the ligands. We monitored the interaction energies of important amino acid side chains during EM and MD. Typical results in case of His90, Arg 120, Tyr355 and Tyr385 are given in Table 1. We also monitored points of contact between inhibitors and amino acid side chains of COX-2, nature and duration of their interactions in regions 1-3 (Table 2).

Regionl NS as also BFI-BF3, because of their smaller

lengths in comparison with the length of the channel (25 A), reside mostly in the lower part of the cavity and do not have many specific interactions in region 1. DHDMBFs interacted only with Glu524 at lower end of this region while NS had hydrophobic interaction with Ala527. BF3 also interacted with Phe381 and Leu384. The maximum number of contacts (six) was seen in case of Mel. It interacted with all the residues in this region except Glu524 and Phe381 (Table 2). Pir also interacted equally well with all the residues except Phe381 and Leu384. Ten could interact only with Tyr385, Trp387, Met522 and backbone of Ala527. In the energy minimized models, maximum interaction of Tyr385 is seen with Mel. Although there are no structure based models for

COX-I and COX-2 activity, interaction of Tyr385 is considered to be significant for SAID's activity. Tyr385 is believed to be involved in stereo-specific removal of pro S hydrogen from arachidonic acid and subsequent insertion of oxygen4. Peroxidase activates COX by radical transfer from feryl oxy porphyrin of intermediate I to a tyrosine 9-12 A away and removes oxygen from arachidonic acid49. Tyr385 also has good contact with Ind.

Region 2 Region 2 is important fo r potency of several

NSAIDs. Arg120 in this region interacts with the carboxylic moieties of the aryl propionic class of inhibitorsI4.15.19.2o. It has been recognized as the

essential requirement for NSAIDs action from a long timeso. However, the current view is lightly di fferent. It is felt that it may not be required for the action of COX-2 selective neutral NSAIDs51. We found a good interaction of this residue in all the ligands except Ten. All the three DHDMBFs interacted well with Arg120. Amongst them, BF3 showed maximum interaction. In case of NS and Ind interaction of Arg120 with COX-l was much stronger. Interaction energies with NS and Ind were - 3.3 and - 5.8 kCalimole respectively in MD models and -9.4 and -7.6 kCal/mole respectively in EM models. This can be understood in the light of the fac t th at in COX-I this is the only charged basic ligand in the active cavity. Our results showed that interaction of Arg 120 is important for the activity of all acidic NSAIDs.

Ind and BFI make series of contacts with the loop adjacent to membrane binding domain (TyrI15 , Va1116, Leu 117, Ser 119 and Ser 121 ). Mel also seems to interact well with large number of residues in the region 2 (Vall 16, Va1349, Leu352 and Tyr355) . All oxicams interacted with Ser530, which is also an important amino acid. Acetylation of Ser530 blocks the channel entri. Pir and Ten also interact with Leu53 1.

Another important residue in this region is Tyr355 which is located at the entrance of the channel. It is implicated by many authors28-3o in the specificity of action of 2-phenylpropionic class of inhibitors. Mutation of this residue to phenylalanine produced stereo-chemical changes but no effect on potenc/9

.

We observed that atom OG of Tyr355 formed H-bond with S02 of NS in NS-COX-2 complex for 50% of the time, while for 30% of the time it was within the limits of electrostatic interaction. NIH of NS was at an average distance of 3.5 A from OG of Tyr355 and

KOTH EKAR el al. : RECOGNITION OF CYCLOOXYGENASE-2 (COX-2) ACTIV E SITE 61

Table 2- Points of contacts of li gands with amino acid side chains in EM model

Residues NS Ind BF1 BF2 BF3 Mel Pir Ten Pept

Region-I

Phe38 I ,.j ,.j

Leu384 ,.j ,.j

Tyr385 ,.j ,.j ,.j ,.j ,.j

Trp387 ~ ,.j ~ ,.j ,.j

Mel522 ,.j ,.j ,.j ,.j

Glu524 ,.j ~ ,.j ,.j ~

Gly526 ,.j ,.j ~

Al a527 ,.j ~ ,.j ~ ,.j ~

Region-2

1Ie11 2 ,.j ,.j

Met1 13 ~

Tyrl15 ,.j ,.j

Vall1 6 ~ ,.j ~ ~

Leul17 ,.j ~

Serl19 ,.j

Arg 120 ,.j ,.j ,.j ~ ~ ,.j ,.j

Serl21 ,.j

Val349 ,.j ~ ,.j

Leu352 ,.j ,.j ,.j ,.j

Ser353 ,.j

Gly354 ,.j ,.j

Tyr355 ,.j ~ ,.j ,.j ,.j ,.j ,.j ~

Phe357 ,.j

Leu359 ,.j ,.j

Ser530 ,.j ~ ,.j ~ ,.j

Leu53 I ~ ,.j ,.j

Region-3

Pro86 ,.j ,.j

Val89 ,.j ,.j ,.j

His90 ,.j ,.j ,.j ,.j ,.j

Leu93 ,.j

Gly94 ,.j

Glnl92 ,.j ,.j

Leu507 ,.j

Arg5J3 ,.j ~ ,.j ,.j ,.j ,.j

Ala51 6 ,.j ,.j

lIe5 J 7 ,.j ,.j ,.j

Phe51 8 ,.j ,.j ,.j ,.j

Val523 ,.j ,.j ,.j

62 INDI AN J. BIOCHEM. I3I OPH YS ., VO L. 38, FEI3 RUAR Y & APRIL 200 1

al so had an electrostati c interacti on with it. In NS­COX- I complex, the backbone NH of T yr3SS was within H-bonding limits of N02 for 10% of the time. It had an electros tati c interac ti on fo r 50% of the time. In case of Ind-COX-2 complex, its O2 group was within H-bonding limit of OG of T yr3SS for 5% of the time. In Ind-COX- I complex, onl y the start ing model had H-bonding interaction with Tyr3SS. The interacti on in case of NS was stronger than with other li gands. Oxicams interacted better than DHDMBFs. The max imum interaction was seen with Mel , the most effecti ve amongst the ox icams.

Region 3 Region 3 is important for COX-2 selectivity. A

non-selective inh ibitor Ind shows no contacts in thi s region. NS, BFl , BF3 and Mel had good interacti on with Hi s90. Interaction energies fo r NS and BFl with Hi s90 are - 5.8 and -4.6 kCalimole respectively and are signi ficantly lower than for other ligands. DHDMBFs interac ted very well with ArgS13, so also did Pir. NS and ox icams interac ted with lieS 17, PheS 18 and Va1S23.

We monitored H-bonding network amongst Arg 120, GluS24, Tyr3SS and ArgS 13 to understand compactness of the channel entrance. We found consistent interaction between Arg 120-G luS24, ArgS 13-GluS24, and Tyr3SS-GluS24 during MD in case of Mel. In Ind and BF3, H-bonding and electrostatic interacti ons were noticed between Arg 120-GluS24 and ArgS 13-GluS24 for 60% of the time. In BFl only a H-bond was seen between Arg 120-GluS24 and in NS, we found a H-bond and an electrostatic interaction between ArgS 13-GluS24 leading to a more relaxed conformation. The hi gher potency of BF3 and longer duration of activity of Mel may be due to compactness at the chan nel entrance. The COX-2 selecti ve li gands, NS and BFl, led to a more relaxed conformation at the channel entrance due to their larger sizes.

The syntheti c peptide des igned by us had a length of 16.5 A which was more than 70% of the channel

• 0 3 length With largest volume (2090 A) and lowest binding energy (-89.96 and -1 22 kCalimole in MD and EM models). It interacted with all t e residues in the active cav ity except Leu384 in the reg ion I, Leull7 , Ser11 9, Serl 2 1 and Gl y3S4 in region 2 and Gly94, LeuS07,lIeSI 7, PheS IS and ValS23 in region 3. We fO Lind H-bond between ArgS 13-GluS24 fo r almost all the peri od and the conformatio n of COX-2 at the ent rance was relaxed, simil ar to NS and BFl COX-2 selective inhibitors.

Summary and Conclusion The resufts presented in this paper show that there is

no simple correlation between interaction energies of the inhibi tors and potency amongst different chemi cal se ri es . Within the same series, occas ionally we find some correlation between binding energies and lCso values. Entropi c contributions in the binding energies, errors in es timating IC50 va lues and different models used for pharmacological experi men ts lead to these di fferences. At best one can compare binding energy differences in two enzymes with re lative IC50 vai ues. In this case most of the errors get cancelled out.

Compari son of inhibitor volumes and interact ion energies of specific side chains of the enzy me with pharmaco-kinetic parameters give a better correlat ion. These interactions depend on overall dimensions and chemical nature of the li gands which are di fficult to estimate usi ng automated modelling packages. Also one needs more inpu ts fro m the pharmacological studi es.

On the basis of the obtained results, we found that the peptide with .'1phe was a sui t bl e inhibitor for COX-2. It did not di stort the enzyme, occupied over 90% cavity volume, had low interac ti on energy as compared to other COX-2 specific and non-specific ligands and made a number of speci fic co ntacts considered to be important for COX-2 acti vity and selectivity. Size-wise, the peptide was too large to be incorporated in the COX-I cavi ty and could be thus, a COX-2 selec ti ve inhibitor. Further work on the design of COX-2 selecti ve peptides is on.

Acknowledgement

The authors are thankful to the Department of Sci­ence and Technology for financial ass istance. SS is thank ful to Council of Scienti fic and Industri al Re­search fo r the award of Sen ior Research Fellowship.

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