Design, Synthesis, and Evaluation of New Tripeptides as COX-2

Hindawi Publishing CorporationJournal of Amino AcidsVolume 2013 Article ID 606282 7 pageshttpdxdoiorg1011552013606282

Research ArticleDesign Synthesis and Evaluation of New Tripeptides asCOX-2 Inhibitors

Ermelinda Vernieri1 Isabel Gomez-Monterrey2 Ciro Milite1 Paolo Grieco2

Simona Musella3 Alessia Bertamino1 Ilaria Scognamiglio4 Stefano Alcaro5 Anna Artese5

Francesco Ortuso5 Ettore Novellino2 Marina Sala1 and Pietro Campiglia1

1 Dipartimento di Scienze Farmaceutiche e Biomediche Universita degli Studi di Salerno Via Ponte don Melillo 84084 Fisciano Italy2 Dipartimento di Chimica Farmaceutica e Tossicologica Universita degli Studi di Napoli Federico II Via D Montesano 4980131 Napoli Italy

3 Dipartimento Farmaco-Biologico Universita degli Studi di Messina Viale Annunziata 98168 Messina Italy4Dipartimento di Biochimica e Biofisica Seconda Universita degli Studi di Napoli Via L De Crecchio 7 80138 Napoli Italy5 Dipartimento di Scienze della Salute Universita degli Studi ldquoMagna Graeligciardquo di Catanzaro Campus Universitario ldquoS VenutardquoViale Europa 88100 Catanzaro Italy

Correspondence should be addressed to Marina Sala msalaunisait

Received 21 December 2012 Accepted 17 January 2013

Academic Editor Michele Caraglia

Copyright copy 2013 Ermelinda Vernieri et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins which aremediatorsof inflammation It exists mainly in two isoforms COX-1 and COX-2 The conventional nonsteroidal anti-inflammatory drugs(NSAIDs) have gastrointestinal side effects because they inhibit both isoforms Recent data demonstrate that the overexpression ofthese enzymes and in particular of cyclooxygenases-2 promotes multiple events involved in tumorigenesis in addition numerousstudies show that the inhibition of cyclooxygenases-2 can delay or prevent certain forms of cancer Agents that inhibit COX-2 whilesparingCOX-1 represent a new attractive therapeutic development and offer a newperspective for a further use of COX-2 inhibitorsThe present study extends the evaluation of the COX activity to all 203 possible natural tripeptide sequences following a rationalapproach consisting in molecular modeling synthesis and biological tests Based on data obtained from virtual screening onlythose peptides with better profile of affinity have been selected and classified into two groups called S and E Our results suggestthat these novel compounds may have potential as structural templates for the design and subsequent development of the newselective COX-2 inhibitors drugs

1 Introduction

The main cause of the inflammation is the prostaglandinsoverproduction which are synthesized by cyclooxygenaseenzymes [1]

Prostaglandin-endoperoxide synthase commonly calledcyclooxygenase (COX) is an intracellular enzyme requiredfor the conversion of arachidonic acid to prostaglandins Thetwo best-known COX isoforms are referred to as COX-1 andCOX-2 for the order in which they were discovered [2] Thefirst isozyme is constitutively expressed in resting cells ofmost tissues functions as a housekeeping enzyme and is

responsible for maintaining homeostasis (gastric and renalintegrity) and normal production of prostaglandins viceversa the COX-2 expression is induced by infection and it isresponsible for the inflammatory response Such a differencesuggested to report COX-1 as the constitutive form andCOX-2 as the inducible one More recently the constitutivepresence of COX-2 has been highlighted in brain kidney andendothelial cells but is virtually absent in most other tissuesIn particular COX-2 expression is significantly upregulatedas part of various acute and chronic inflammatory conditionsand in neoplastic tissues The design of a selective inhibitoris difficult as the COX-1 and COX-2 binding sites are almost

2 Journal of Amino Acids

identical and the isoforms show sequence homology of 60ndash65 Experimental 3D models of both enzyme structureshave shown the complexity of the problemThey suggest thatthe tertiary conformations of these proteins are very similarand the substrate binding pocket and catalytic site aminoacids are nearly identical in both enzymes In this case theonly difference is the substitution at residue position 523 ofthe COX-1 Ile by the COX-2 Val that opening an additionalpocket results in an enzyme inducible form binding sitelarger than the constitutive one The structural similaritiesof COX-1 and COX-2 enzymes have made the developmentof selective inhibitors for COX-2 versus COX-1 a specialchallenge Since the discovery of the COX-2 enzyme in theearly 1990s numerous COX-2 selective inhibitors have beenproposed (Figure 1)

A common structural feature of these selective COX-2inhibitors is the presence of two vicinal aryl rings attached toa central five- or six-membered heterocyclic or carbocyclicmotif Typical examples of selective COX-2 inhibitors likecelecoxib rofecoxib valdecoxib etoricoxib and SC57666demonstrate that a broad variety of five- or six-memberedcarbo- and heterocycles are acceptable for binding to thecyclooxygenase active site

Recent reviews on the current status of COX-2 inhibitorsfurther confirm the flexibility of the carbocyclicheterocycliccoremotif for COX-2 binding [3] Good results were obtainedwith the coxib family but their secondary effects especiallyaffecting the kidney and central nervous system stimulatedthe research towards the most powerful substances with alower isoform selectivity On the basis of the previouslyreported information the present study extends the evalua-tion of the COX activity to all 203 possible natural tripeptidesequences following a rational approach consisting in molec-ular modeling synthesis and biological tests

2 Materials and Methods

21MolecularModeling ThePDBentries 1Q4G [4] and 1PXX[5] have been considered as COX-1 and COX-2 receptormodels respectivelyThe crystallographic resolution equal to20 A for 1Q4G and to 29 A for 1PXX and the kind of cocrys-tallized ligands two reversible NSAIDs have been the choicecriterion The original ligands 120572-methyl-4-biphenylaceticacid and diclofenac respectively have been removed andhydrogen atoms missing into the PDB files have been addedby means of the Maestro GUI [6] In order to appropriatelytake into account structural solvent all cocrystallized watermolecules within 5 A from the protein atoms have beenincluded in our preliminary models Added hydrogen atomsposition has been optimized according to MMFFs [7ndash10]force field implemented in MacroModel ver 72 [11] Theprocedure consisted in a preliminary energy minimizationcomputed applying to all nonhydrogen atoms a constantforce equal to 200KJmolsdotA followed by a further watermolecules unconstrained run Using the same force field andthe same constraints both receptor models final structureshave been submitted to 500 ps of molecular dynamics at300∘K with an integration time step equal to 15 fs In all

simulation a distance-dependent dielectric constant equals to4 has been adopted Such an approach highlighted the mosttightly interacting water molecules that maintained theirposition while the other ones have been moved far from thereceptor models In order to achieve a fully relaxed confor-mation resulting molecular dynamics structures containingthe tightly interacting solvent molecules only have beensubmitted to unconstrained energy minimization producingour COX-1 and COX-2 receptor models

The 3D tripeptides library was built by an in-housePerlMol Chemistry modules-based software All possiblecombination of 20 the (L)-series natural amino acids havebeen considered and the resulting 8000 tripeptide modelswere energy minimized using the same force field reportedfor the receptors

The docking binding site for both targets has beendefined by means of a regular box centered onto the proteinchain A Tyr 385 Its volume was about 390000 A3 widelycovering COX-1 and -2 binding sites Glide grid maps havebeen computed using the standard precision algorithm Ouroptimized library has been submitted to flexible Glide dock-ing simulation with respect to both COX receptor modelsreducing the ligands van der Waals atom radius by a 08factor Default Glide interaction energies (ECvdW) have beenadopted for scoring the tripeptide docking poses

22Materials 119873120572-Fmoc-protected amino acidsWang resinHOBt HBTU DIEA piperidine and trifluoroacetic acidwere purchased from Iris Biotech (Germany) Peptide syn-thesis solvents reagents and CH

3CN for HPLC were reagent

grade and were acquired from commercial sources (SigmaAldrich Italy) and used without further purification unlessotherwise noted

23 Synthesis The synthesis of tripeptides (S1ndashE10) was per-formed according to the solid phase approach using standardFmoc methodology in a manual reaction vessel [12] The firstamino acid N120572Fmoc-Xaa-OH was linked onto the Wangresin (100ndash200mesh 1 DVB 11mmolg) and was attachedto Wang resin using HOBtHBTU as an activating agent(3eq) and a catalytic amount of DMAP

The following protected amino acids were then addedstepwise N120572-Fmoc-Met-OH N120572-Fmoc-Arg(Pbf)-OH N120572-Fmoc-His(119873

(119894119898)trityl (Trt))-OH (or N120572-Fmoc-Gln(Trt)-OH)

N120572-Fmoc-Gly-OH N120572-Fmoc-Trp(Boc)-OH N120572-Fmoc-Glu(OtBu-OH N120572-Fmoc-Asp(OtBu)-OH N120572-Fmoc-Phe-OH N120572-Fmoc-Lys(Boc)-OH N120572-Fmoc-Ala-OH N120572-Fmoc-Ser(tBu)-OH and N120572-Fmoc-Ile-OH Each coupling reactionwas accomplished using a 3-fold excess of amino acid withHBTU and HOBt in the presence of DIEA (6 eq) TheN120572-Fmoc protecting groups were removed by treating theprotected peptide resin with a 25 solution of piperidinein DMF (1 times 5min and 1 times 25min) The peptide resin waswashed three times with DMF and the next coupling stepwas initiated in a stepwise manner The peptide resin waswashed with DCM (3times) DMF (3times) and DCM (3times) and thedeprotection protocol was repeated after each coupling step

Journal of Amino Acids 3

H3C

H3C

NN

N N

N

OO O

F

MeOS2MeOS2

SO2NH2

CF3

MeO2S

MeO2SCl

Celecoxib Rofecoxib Valdecoxib

Etoricoxib SC57666

Figure 1 Chemical structures of selective COX-2 inhibitors

In addition after each step of deprotection and aftereach coupling step Kaiser test was performed to confirm thecomplete removal of the Fmoc protecting group respectivelyand to verify that complete coupling has occurred on all thefree amines on the resin

The N-terminal Fmoc group was removed as describedabove and the peptide was released from the resin withTFAiPr

3SiHH

2O (90 5 5) for 3 h The resin was removed

by filtration and the crude peptide was recovered by precipi-tation with cold anhydrous ethyl ether to give a white powderand then lyophilized

24 Purification and Characterization All crude peptideswere purified by RP-HPLC on a semipreparative C18-bondedsilica column (Phenomenex Jupiter 250 times 10mm) using aShimadzu SPD 10A UVVIS detector with detection at 215and 254 nm

The column was perfused at a flow rate of 3mLmin withsolvent A (10 vv water in 01 aqueous TFA) and a lineargradient from 10 to 90 of solvent B (80 vv acetonitrilein 01 aqueous TFA) over 40min was adopted for peptideelution Analytical purity and retention time (tR) of eachpeptidewere determined usingHPLC conditions in the abovesolvent system (solvents A and B) programmed at a flow rateof 1mLmin using a linear gradient from 10 to 90 B over25min fitted with C-18 column Phenomenex Jupiter C-18column (250 times 460mm 5 120583)

All analogues showed gt97 purity when monitored at215 nm Homogeneous fractions as established using analyt-ical HPLC were pooled and lyophilized

Peptides molecular weights were determined by ESImass spectrometry ESI-MS analysis in positive ion modewere made using a Finnigan LCQ ion trap instrumentmanufactured by Thermo Finnigan (San Jose CA USA)

Table 1 Structures and analytical data of tripeptides synthesized

Peptide Structure HPLC ESI-MS YieldtR found calc

S1 GMD 1050 32208 32110 75S2 ERA 999 3753 37419 80 S3 GHE 808 34214 34113 67S4 GER 1000 36117 36018 80S5 DRC 989 39320 39215 62S6 ARA 1046 31724 31619 68S7 PER 887 40140 40021 81S8 KHI 1098 39711 39625 80S9 AER 1100 37532 37419 75S10 AGR 903 30334 30217 79E1 SRH 895 39930 39820 68E2 SWE 804 4210 42016 69E3 IRT 803 3893 38824 76E4 SMD 800 34216 35111 78E5 GRN 843 3462 34518 65E6 SHE 868 37217 37114 76E7 SQE 845 36317 36214 67E8 SMH 946 37426 37314 75E9 ARM 803 3776 37619 77E10 AQE 997 3470 34615 76tR peptide retention time

equipped with the Excalibur software for processing the dataacquiredThe sample was dissolved in a mixture of water andmethanol (5050) and injected directly into the electrospraysource using a syringe pump which maintains constant flowat 5 120583LminThe temperature of the capillary was set at 220∘C(Table 1)


25 Biological Assay

251 Preparation of Washed Platelets Washed human plate-lets were prepared from blood anticoagulated with citrate-phosphate-dextrose which was obtained from Centro deTransfusion de Galicia (Santiago de Compostela Spain)

Bags containing buffy coat from individual donors werediluted with the same volume of washing buffer (NaCl120mM KCl 5mM trisodium citrate 12mM glucose10mM sucrose 125mM pH 6) and centrifuged at 400 gfor 9min The upper layer containing platelets (platelet-rich plasma) was removed and centrifuged at 1000 g for18min The resulting platelet pellet was recovered resus-pended with washing buffer and centrifuged again at 1000 gfor 15min Finally the platelet pellet from this step wasresuspended in a modified Tyrode-HEPES buffer (HEPES10mM NaCl 140mM KCl 3mM MgCl

205mM NaHCO

3

5mM glucose 10mM pH 74) to afford a cell densityof 3ndash35sdot108 plateletmL The calcium concentration in theextracellular medium was 2mM [13]

252 hCOX Activity The potential effects of the test drugson total hCOX activity (bis-dioxygenase and peroxidasereactions) were investigated by measuring their effects on theoxidation of NNN1015840N1015840-tetramethyl-p-phenylenediamine(TMPD) to NNN1015840N1015840-tetramethyl-p-phenylenedaimineusing AA as common substrate for both hCOX-1 and hCOX-2 microsomal COX-2 prepared from insect cells (Sf21cells) infected with recombinant baculovirus containingcDNA inserts for hCOX-2 (Sigma Aldrich Quımica SAAlcobendas Spain) and COX-1 from human plateletmicrosomes (obtained as described in the above paragraphsince unlike hCOX-2 hCOX-1 is not available commercially)[14]

3 Results and Discussion

31 Design The purpose of this work consists in the identifi-cation of new peptide ligands for COX that show comparedto the current state of the art a greater power a lower toxicityand a high average degree of selectivity

In particular the attention has been focused on cyclooxy-genase 2 (COX-2) as it has been recently shown to promotemultiple events in the tumorigenesis process [15] Severalreports indicate that COXs inhibitors can prevent the devel-opment of various human tumors including colon breastlung liver and gastric neoplasias [16ndash20]

For several types of cancer the real risk factor seems tobe chronic inflammation that maintains a high level of COX-2 and increases events that promote tumor formation Atragic example of this mechanism ismalignantmesothelioma(MM) a rare tumor of the mesothelial surface of the pleuraland peritoneal cavities [21]

The aim of this study is to demonstrate the possibilityof modulation of the activity of COX through peptides thatmay be found in sites in which the inflammatory process is inplace

To achieve this goal we relied on the data reported in theliterature focused the attention on the structure of COX-2One of the known potent inhibitors of COX-2 is SC-558 (adyaril heterocyclic inhibitor) which contains a bromophenylring a pyrazole group and a phenylsulphonamide moietyMost of the sulphur containing NSAIDs are selective COX-2 inhibitors and this sulphur atom reduces the toxicity of thecompound

Using this information in a recent work Somvanshi et alhave designed and synthesized several tripeptide sequencescontaining a hydrophobic amino acid with aromatic ring acysteine residue which contains sulphur atom and a chargedresidue at the C-terminal end [1] In particular 15 tripeptideswere screened by ELISA test and the best of them tripeptideWCS inhibited more than 85 of the COX-2 activity

Though the chemical nature of sulphur atom of sulphon-amide group in SC-558 and in cysteine residue is differentstill similarities in binding constant of peptide with knownNSAIDs SC-558were observedThus preventing the reactionof substrate arachidonic acid with the enzyme supportsthe possibility of peptide WCS as potent and competitiveinhibitor of COX-2 As the phenyl ring of SC-558 interactswith residues in hydrophobic cavity of COX-2 formed by Phe381 Leu 384 Tyr 385 Trp 387 Phe 518 and Ser 530 it canbe assumed that the aromatic ring of tryptophan residue ofpeptide will also interact with residues in hydrophobic cavityThe free carboxylate group of the peptide can electrostaticallyinteract to Arg 120WCS can be considered as a potential leadcompound for developing a new class of COX-2 inhibitors

Extending the study of Somvanshi et al [1] we havebuilt a complete tripeptide virtual library containing allpossible combinations of the 20 (L)-series natural aminoacids The COX-1 and -2 binding pocket recognition ofthe 8000 library hits has been investigated by means ofmolecular docking techniques and the resulting complexesstability has been evaluated using the theoretical ligand-enzyme binding energies The selection of the peptides forthe experimental tests has been driven by their affinity withrespect to both COX isoforms Such a task has been carriedout by computing the ratio between COX-2 and COX-1peptide interaction energy The top two COX-2 interactingcompounds still maintaining affinity for the COX-1 and onetheoretically inactive peptide have been selected The firstcouple corresponds to the sequences Gly-Met-Asp (GMD)and Gly-His-Glu (GHE) while the last one is Tyr-Tyr-Val(YYV) GMDandGHE showed a strong recognition toCOX-2 and a weaker interaction to COX-1 YYV was unable to fitinto both binding pockets due to its steric hindrance (Table 2)

The graphical inspection of the GMD and GHE COXscomplexes revealed for both peptides remarkable differentrecognition of the two enzymes (Figure 2)

In all cases our compounds have occupied the knownNSAID binding pocket Interestingly the number of enzymeinteracting residues comprise between 21 and 26 is quitesimilar but the COX-2 hydrogen bond network is muchbetter than COX-1 and could explain the selectivity of ourligands COX-2 Ser 530 side chain is involved in hydrogenbond to GMD backbone and through a water moleculebridge to the carboxylate groups of both our active peptides


Figure 2 COX-1 (a) and (b) and COX-2 (c) and (d) recognition of GMD and GHE Tripeptides are depicted in polytube CPK colorednotation interacting enzyme residues in green carbons wireframes HEME cofactor in green carbons spacefill and the rest of the enzyme isshowed in transparent green cartoon Yellow dotted lines indicated hydrogen bond interaction

Table 2 COXs theoretical binding energies (be) in kcalmolnumber of van der Waals interacting enzyme residues (ir) andintermolecular hydrogen bonds (hb)

Peptide COX-1 COX-2be ir hb be ir hb

GMD minus120 23 1 minus3987 21 3GHE minus056 25 0 minus4215 26 5YYV mdash mdash mdash mdash mdash mdash

Tripeptide carboxylate moieties are also involved in hydro-gen bonds to COX-2 catalytic Tyr 385 GHE still reports suchan interaction between its protonated N-terminal and Leu352 backbone Even if GMD through its C-terminal showsone hydrogen bond to Tyr 355 and favorable electrostaticinteraction to COX-1 Arg 120 the steric hindrance of COX-1 Ile 523 bulkier than COX-2 Val limits the enzyme cleftrecognition preventing our compounds in particular GHEfrom establishing hydrogen bonds highlighted inCOX-2Theremaining part of contribution to the COXs recognition ofour peptides could be addressed an unspecific van der Waalsinteraction

At the same time based on data obtained from virtualscreening only those peptides with better profile of affinityhave been selected and classified into two groups called Sand E (Table 1) The first group includes 10 sequences capableof interacting with both enzymatic isoform but with a clearpreference towards COX-2 In the second group are placedfurther 10 tripeptides which have shown affinity exclusivelyfor COX-2 and no reconnaissance towards COX-1

32 Chemistry All peptides S1-E10 were synthesized by stan-dard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry usingan appropriate orthogonal protection strategy Peptides werereleased from the solid support using a cleavage cocktail of90 TFA 5 water and 5 Et

3SiH All analogues showed

gt97 purity at HPLC analysis

33 Anti-infiammatory Activity The biological assays werecarried out to evaluate the inhibitory activity against COX-2 by the group of Professor Dr Francisco Orallo Departmentof Pharmacology Faculty of PharmacyUniversity of Santiagode Compostela Spain

Results shown in Table 3 are expressed as means plusmn SEMfrom five experiments Means were compared by one-way


Table 3 Inhibitory activity of compounds synthesized and selectiv-ity against COX-2 over COX-1

COX-1 (IC50) COX-2 (IC50) RatioIndometacina 1216 plusmn 116120583M 3520 plusmn 141 120583M 29Diclofenac 1823 plusmn 173120583M 2362 plusmn 197 120583M 13FR122047 9380 plusmn 655 120583M lowastlowastlowast

gt1066a

Nimesulide lowastlowastlowast 23140 plusmn 1984 120583M lt046a

DuP697 2261 plusmn 156 120583M 12632 plusmn 741 120583M 00056S1 15033 plusmn 234 120583M 9404 plusmn 259 120583M 06255S2 14321 plusmn 257 120583M 12092 plusmn 233 120583M 08443S3 15244 plusmn 518 120583M 9489 plusmn 212120583M 06225S4 9932 plusmn 114120583M 8056 plusmn 214 120583M 08111S5 16143 plusmn 257 120583M 10001 plusmn 233 120583M 06195S6 10231 plusmn 114120583M 9120 plusmn 241 120583M 08914S7 10033 plusmn 219 120583M 8821 plusmn 301 120583M 08792S8 12248 plusmn 378 120583M 9166 plusmn 298 120583M 07484S9 22157 plusmn 104120583M 6834 plusmn 543 120583M 0308S10 9911 plusmn 155 120583M 7920 plusmn 215120583M 07991E1ndashE10lowastlowast mdash mdash mdashSignificant differences between the two means (P lt 005 or P lt 001)were determined by one-way analysis of variability (ANOVA) followed byDunnettrsquos post hoc testlowastlowastlowastNo active at 500120583M (the highest concentration tested)aValue obtained whereas the corresponding IC50 to COX-1 or COX-2 is thehighest concentration testedlowastlowastData not shown

analysis of variance (ANOVA) followed by Dunnettrsquos post-hoc test The inhibitory effects of the tested compounds areexpressed as IC50 (concentrations that produce reduction of50 of the enzymatic activity of COX control isoform) esti-mated by least-squares linear regression using the programOrigin 50 with X = log molar concentration of the testedcompounds and Y = of pharmacological response

This regression was performed using the data obtainedwith 4ndash6 different concentrations of each compound assayedwhich inhibited the enzymatic activity of COX controlisoform between 20 and 80

Finally they were calculated the corresponding indices ofselectivity (SI) of COX-1 SI = [IC

50(COX-2)][IC

50(COX-1)]

As demonstrated by the virtual screening all twentytripeptides show a greater selectivity against COX-2 overCOX-1

In particular peptide S9 shows a very interesting profileof both selectivity and inhibitory potency towards COX-2 infact the selectivity index between COX-2 andCOX-1 is about0308 more selective than the nimesulide that has an indexof about 046 moreover this peptide also shows an increasein activity compared to the same drug (6834 plusmn 543 120583M S9activity 23140 plusmn 1984 120583M nimesulide activity)

Analyzing biological data depending both on the chem-ical structure that the values of the energies of binding thepeptides S9 S10 S7 and S4 show an analogous biologicalprofile (selectivity and affinity)

However a complete analysis of the structure-activityrelationship of these peptides cannot be performed because ofthe small number of peptides that limit the goodness of this

report It is possible to highlight two important aspects theguanidine group of Arg at C-terminal and the carboxyl groupin the side chain of the second amino acid are requirementsfor the interaction with the target while all peptides that havea carboxyl group in the side chain on the first amino acidshow a loss of selectivity that of affinity the aromatic grouppresent in WCS peptide lead is not essential to interact withthe target

In conclusion previously reported peptides seem toreflect too high potency and selectivity instead peptides ofseries E do not result selective for COX-2 (data not shown)Further studies for the peptides E1ndashE10 are in progress

4 Conclusion

There is an increasing interest in the development of newtreatments based on cyclooxygenases-2 inhibitors to prolongsurvival and even potentially cure various forms of cancer asmalignant mesothelioma

The present study describes hit identification synthesisand biological evaluation of a series of linear tripeptidesmostof them are able to selectively inhibit COX-2 Further otherexperiments aimed to verify the potentiality of these peptidesas anticarcinogenic drugs as well as the preparation of novelmore potent and selective peptidomimetic derivatives are inprogress

Abbreviations used for amino acids follow the rules of theIUPAC-IUB Commission of Biochemical Nomenclature in JBiol Chem 1972 247 977-983 Amino acid symbols denoteL-configuration

Abbreviations

DMAP 4-dimethylamino-pyridineTFA Trifluoroacetic acidDCM DichloromethaneDIPEA NN-diisopropylethylamineDMF NN-dimethylformamideEt3SiH Triethylsilane

Fmoc 9-fluorenyl-methoxycarbonylHOBt N-hydroxy-benzotriazoleHBTU 2-(1H-benzotriazole-1-yl)-1133-

tetramethyluroniumhexafluoro-phosphate

RP-HPLC Reversed-phase high performance liquidchromatography

ESI Electrospray ionizationLC-MS Liquid chromatography-mass

spectrometryPbf 22467-pentamethyldihydrobenzofuran-

5-sulfonyl

Disclosure

Theauthors of the paper do not have a direct financial relationwith the commercial identity mentioned in thier submittedpaper that might lead to a conflict of interests for any of theauthors


Acknowledgments

The assistance of the staff is gratefully appreciated DrFrancisco Orallo died unexpectedly on December 9 2009The authors dedicate this work to his memory

References

[1] R K Somvanshi A Kumar S Kant et al ldquoSurface plasmonresonance studies and biochemical evaluation of a potent pep-tide inhibitor against cyclooxygenase-2 as an anti-inflammatoryagentrdquo Biochemical and Biophysical Research Communicationsvol 361 no 1 pp 37ndash42 2007

[2] M E Turini and R N DuBois ldquoCYCLOOXYGENASE-2 atherapeutic targetrdquo Annual Review of Medicine vol 53 pp 35ndash57 2002

[3] S K Sharma B J Al-Hourani M Wuest et al ldquoSynthesis andevaluation of fluorobenzoylated di- and tripeptides as inhibitorsof cyclooxygenase-2 (COX-2)rdquo Bioorganic amp Medicinal Chem-istry vol 20 pp 2221ndash2226 2012

[4] K Gupta B S Selinsky C J Kaub A K Katz and P J Loll ldquoThe20 A resolution crystal structure of prostaglandinH2 synthase-1 structural insights into an unusual peroxidaserdquo Journal ofMolecular Biology vol 335 no 2 pp 503ndash518 2004

[5] S W Rowlinson J R Kiefer J J Prusakiewicz et al ldquoAnovel mechanism of cyclooxygenase-2 inhibition involvinginteractions with Ser-530 and Tyr-385rdquo Journal of BiologicalChemistry vol 278 no 46 pp 45763ndash45769 2003

[6] Schrodinger LLC New York NY USA 2010[7] T A Halgren ldquoMerck molecular force field I Basis form

scope parameterization and performance of MMFF9rdquo Journalof Computational Chemistry vol 17 pp 490ndash519 1996

[8] T A Halgren ldquoMerck molecular force field II MMFF94van der Waals and electrostatic parameters for intermolecularinteractionsrdquo Journal of Computational Chemistry vol 17 pp520ndash552 1996

[9] T A Halgren ldquoMMFF VI MMFF94s Option for energyminimization studiesrdquo Journal of Computational Chemistry vol20 pp 720ndash729 1999

[10] T A Halgren ldquoMMFF VII Characterization of MMFF94MMFF94s and other widely available force fields for confor-mational energies and for intermolecular-interaction energiesand geometriesrdquo Journal of Computational Chemistry vol 20pp 730ndash748 1999

[11] F Mohamadi N G J Richards W C Guida et alldquoMacroModel-An integrated software system for modelingorganic and bioorganic molecules using molecular mechanicsrdquoJournal of Computational Chemistry vol 11 pp 440ndash467 1990

[12] E Atherton and R C Sheppard Solid-Phase Peptide SynthesisA Practical Approach IRL Oxford UK 1989

[13] S Vilar E Quezada L Santana et al ldquoDesign synthe-sis and vasorelaxant and platelet antiaggregatory activitiesof coumarin-resveratrol hybridsrdquo Bioorganic and MedicinalChemistry Letters vol 16 no 2 pp 257ndash261 2006

[14] R Fioravanti A Bolasco F Manna et al ldquoSynthesis and bio-logical evaluation of N-substituted-3 5-diphenyl-2-pyrazolinederivatives as cyclooxygenase (COX-2) inhibitorsrdquo EuropeanJournal of Medicinal Chemistry vol 45 pp e6135ndashe6138 2010

[15] E P Spugnini G Citro and A Baldi ldquoCox inhibitors aspotential chemotherapic drugs for mesotheliomardquo CurrentRespiratory Medicine Reviews vol 3 no 1 pp 15ndash18 2007

[16] F A Sinicrope ldquoTargeting cyclooxygenase-2 for prevention andtherapy of colorectal cancerrdquoMolecular Carcinogenesis vol 45no 6 pp 447ndash454 2006

[17] J B Meric S Rottey K Olaussen et al ldquoCyclooxygenase-2 asa target for anticancer drug developmentrdquo Critical Reviews inOncologyHematology vol 59 no 1 pp 51ndash64 2006

[18] D Mazhar R Ang and J Waxman ldquoCOX inhibitors and breastcancerrdquo British Journal of Cancer vol 94 no 3 pp 346ndash3502006

[19] S Ali B F El-Rayes F H Sarkar and P A Philip ldquoSimul-taneous targeting of the epidermal growth factor receptorand cyclooxygenase-2 pathways for pancreatic cancer therapyrdquoMolecular Cancer Therapeutics vol 4 no 12 pp 1943ndash19512005

[20] TWu ldquoCyclooxygenase-2 in hepatocellular carcinomardquoCancerTreatment Review vol 32 pp 28ndash44 2006

[21] I Cardillo E P Spugnini A Verdina R Galati G Citro andA Baldi ldquoCox and mesothelioma an overviewrdquo Histology andHistopathology vol 20 no 4 pp 1267ndash1274 2005

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identical and the isoforms show sequence homology of 60ndash65 Experimental 3D models of both enzyme structureshave shown the complexity of the problemThey suggest thatthe tertiary conformations of these proteins are very similarand the substrate binding pocket and catalytic site aminoacids are nearly identical in both enzymes In this case theonly difference is the substitution at residue position 523 ofthe COX-1 Ile by the COX-2 Val that opening an additionalpocket results in an enzyme inducible form binding sitelarger than the constitutive one The structural similaritiesof COX-1 and COX-2 enzymes have made the developmentof selective inhibitors for COX-2 versus COX-1 a specialchallenge Since the discovery of the COX-2 enzyme in theearly 1990s numerous COX-2 selective inhibitors have beenproposed (Figure 1)

A common structural feature of these selective COX-2inhibitors is the presence of two vicinal aryl rings attached toa central five- or six-membered heterocyclic or carbocyclicmotif Typical examples of selective COX-2 inhibitors likecelecoxib rofecoxib valdecoxib etoricoxib and SC57666demonstrate that a broad variety of five- or six-memberedcarbo- and heterocycles are acceptable for binding to thecyclooxygenase active site

Recent reviews on the current status of COX-2 inhibitorsfurther confirm the flexibility of the carbocyclicheterocycliccoremotif for COX-2 binding [3] Good results were obtainedwith the coxib family but their secondary effects especiallyaffecting the kidney and central nervous system stimulatedthe research towards the most powerful substances with alower isoform selectivity On the basis of the previouslyreported information the present study extends the evalua-tion of the COX activity to all 203 possible natural tripeptidesequences following a rational approach consisting in molec-ular modeling synthesis and biological tests

2 Materials and Methods

21MolecularModeling ThePDBentries 1Q4G [4] and 1PXX[5] have been considered as COX-1 and COX-2 receptormodels respectivelyThe crystallographic resolution equal to20 A for 1Q4G and to 29 A for 1PXX and the kind of cocrys-tallized ligands two reversible NSAIDs have been the choicecriterion The original ligands 120572-methyl-4-biphenylaceticacid and diclofenac respectively have been removed andhydrogen atoms missing into the PDB files have been addedby means of the Maestro GUI [6] In order to appropriatelytake into account structural solvent all cocrystallized watermolecules within 5 A from the protein atoms have beenincluded in our preliminary models Added hydrogen atomsposition has been optimized according to MMFFs [7ndash10]force field implemented in MacroModel ver 72 [11] Theprocedure consisted in a preliminary energy minimizationcomputed applying to all nonhydrogen atoms a constantforce equal to 200KJmolsdotA followed by a further watermolecules unconstrained run Using the same force field andthe same constraints both receptor models final structureshave been submitted to 500 ps of molecular dynamics at300∘K with an integration time step equal to 15 fs In all

simulation a distance-dependent dielectric constant equals to4 has been adopted Such an approach highlighted the mosttightly interacting water molecules that maintained theirposition while the other ones have been moved far from thereceptor models In order to achieve a fully relaxed confor-mation resulting molecular dynamics structures containingthe tightly interacting solvent molecules only have beensubmitted to unconstrained energy minimization producingour COX-1 and COX-2 receptor models

The 3D tripeptides library was built by an in-housePerlMol Chemistry modules-based software All possiblecombination of 20 the (L)-series natural amino acids havebeen considered and the resulting 8000 tripeptide modelswere energy minimized using the same force field reportedfor the receptors

The docking binding site for both targets has beendefined by means of a regular box centered onto the proteinchain A Tyr 385 Its volume was about 390000 A3 widelycovering COX-1 and -2 binding sites Glide grid maps havebeen computed using the standard precision algorithm Ouroptimized library has been submitted to flexible Glide dock-ing simulation with respect to both COX receptor modelsreducing the ligands van der Waals atom radius by a 08factor Default Glide interaction energies (ECvdW) have beenadopted for scoring the tripeptide docking poses

22Materials 119873120572-Fmoc-protected amino acidsWang resinHOBt HBTU DIEA piperidine and trifluoroacetic acidwere purchased from Iris Biotech (Germany) Peptide syn-thesis solvents reagents and CH

3CN for HPLC were reagent

grade and were acquired from commercial sources (SigmaAldrich Italy) and used without further purification unlessotherwise noted

23 Synthesis The synthesis of tripeptides (S1ndashE10) was per-formed according to the solid phase approach using standardFmoc methodology in a manual reaction vessel [12] The firstamino acid N120572Fmoc-Xaa-OH was linked onto the Wangresin (100ndash200mesh 1 DVB 11mmolg) and was attachedto Wang resin using HOBtHBTU as an activating agent(3eq) and a catalytic amount of DMAP

The following protected amino acids were then addedstepwise N120572-Fmoc-Met-OH N120572-Fmoc-Arg(Pbf)-OH N120572-Fmoc-His(119873

(119894119898)trityl (Trt))-OH (or N120572-Fmoc-Gln(Trt)-OH)

N120572-Fmoc-Gly-OH N120572-Fmoc-Trp(Boc)-OH N120572-Fmoc-Glu(OtBu-OH N120572-Fmoc-Asp(OtBu)-OH N120572-Fmoc-Phe-OH N120572-Fmoc-Lys(Boc)-OH N120572-Fmoc-Ala-OH N120572-Fmoc-Ser(tBu)-OH and N120572-Fmoc-Ile-OH Each coupling reactionwas accomplished using a 3-fold excess of amino acid withHBTU and HOBt in the presence of DIEA (6 eq) TheN120572-Fmoc protecting groups were removed by treating theprotected peptide resin with a 25 solution of piperidinein DMF (1 times 5min and 1 times 25min) The peptide resin waswashed three times with DMF and the next coupling stepwas initiated in a stepwise manner The peptide resin waswashed with DCM (3times) DMF (3times) and DCM (3times) and thedeprotection protocol was repeated after each coupling step


H3C

H3C

NN

N N

N

OO O

F

MeOS2MeOS2

SO2NH2

CF3

MeO2S

MeO2SCl


Etoricoxib SC57666




3SiHH












25 Biological Assay



205mM NaHCO

3





























gt1066a






50(COX-2)][IC

50(COX-1)]







4 Conclusion




Abbreviations







5-sulfonyl

Disclosure



Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


H3C

H3C

NN

N N

N

OO O

F

MeOS2MeOS2

SO2NH2

CF3

MeO2S

MeO2SCl


Etoricoxib SC57666




3SiHH












25 Biological Assay



205mM NaHCO

3





























gt1066a






50(COX-2)][IC

50(COX-1)]







4 Conclusion




Abbreviations







5-sulfonyl

Disclosure



Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25 Biological Assay



205mM NaHCO

3





























gt1066a






50(COX-2)][IC

50(COX-1)]







4 Conclusion




Abbreviations







5-sulfonyl

Disclosure



Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















gt1066a






50(COX-2)][IC

50(COX-1)]







4 Conclusion




Abbreviations







5-sulfonyl

Disclosure



Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




gt1066a






50(COX-2)][IC

50(COX-1)]







4 Conclusion




Abbreviations







5-sulfonyl

Disclosure



Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Design, Synthesis, and Evaluation of New Tripeptides as COX-2