Ligand Binding Analysis for Human r5â1Integrin: Strategies for Designing Newr5â1 Integrin Antagonists

Luciana Marinelli,‡ Axel Meyer,†Dominik Heckmann,† Antonio Lavecchia,‡Ettore Novellino,‡ and Horst Kessler*,†

Department Chemie, Technische Universitat Munchen,Lichtenbergstrasse 4, D-85747 Garching, Germany, andDipartimento di Chimica Farmaceutica e Tossicologica,

Universita di Napoli “Federico II”, Via D. Montesano,49-80131 Napoli, Italy

Received December 29, 2004

Abstract: We report a three-dimensional model of the R5â1integrin headgroup bound to the most potent and selectiveligand (SJ749) known to date. The model was built using thecomparative protein modeling method, and it is consistent withexperimental data. From this study, we identified two poten-tially important regions in the R5â1 receptor that are peculiarto this integrin and might be worth considering for drugtargeting.

Integrins are ubiquitous cell adhesion receptors thatbind ligands on the surface of other cells and in theextracellular matrix and are involved in bidirectionalsignaling across the plasma membrane, regulating celladhesion, differentiation, migration, grow, and sur-vival.1 Evidence exists that such receptors are linkedto pathological conditions including tumor progression,thrombosis, immune dysfunction, inflammation, andosteoporosis, so integrins have been attractive thera-peutic targets for several diseases.2,3 Integrin R5â1 andespecially Rv integrins have attracted attention astargets for antiangiogenic therapy.4 Brooks et al. havereported that various low molecular weight ligands,which are recognized by Rvâ3 and Rvâ5 integrins, blockangiogenesis in response to growth factors in tumorsand suppress the cancer growth and metastasis.5 Onthe basis of these findings, the Rvâ3 receptor has beenthe main integrin target in the search for new antican-cer drugs in the past 2 decades, and significant progresshas been made in the identification of selective andpotent Rvâ3 integrin antagonists.6 As a result of ourresearch, the cyclic peptide cyclo(-RGDf[NMe]V-),known as cilengitide, is now in phase II clinical trialsfor patients with glioblastoma.7,14

Nonetheless, recent evidence that mice lacking of â3or â5 integrins exhibit in some cases enhanced angio-genesis8 led to a reevaluation of Rvâ3 and Rvâ5 integrinfunction in angiogenesis.9 The different hypothesis madein an effort to explain the discrepancy between thegenetic results and those obtained using low-molecular-weight ligands are not fully convincing, and the debateis still open. Reconsidering each integrin as a regulatorof angiogenesis, an important issue came out. The R5â1receptor is the only unambiguously proangiogenic in-tegrin; genetic ablation experiments and pharmacologi-

cal results are consistent and strongly support itsimportance in neovascularization processes.10 Hence,the R5â1 integrin is expected to move into the forefrontof research for new effective anticancer drugs.

The research of R5â1 antagonists is not as advancedas that of Rvâ3, and only few ligands are known to bindthe R5â1 integrin.11 This constitutes a considerableobstacle to the rational ligand-based drug design.Moreover, the 3D structure of membrane spanningreceptors such as R5â1 is hard to obtain through X-raycrystallography or NMR methods. Consequently, fewdetailed structural information about ligand-receptorinteractions have been obtained until now. However, itis well-know that integrins, which are heterodimers ofdifferent combinations of noncovalently bound R and âchains, share extensive structural homology. It was alsodemonstrated that the ligand binding to Rv, R5â1, andRIIbâ3 integrins is mediated through the Arg-Gly-Asp(RGD) recognition motif.12,13

A first glimpse at the binding between integrins andthe RGD tripeptide sequence was provided by thecrystal structure13 of the extracellular domain of Rvâ3integrin in complex with cyclo(-RGDf[NMe]V-).14 Ex-periments using X-ray scattering and single-particleelectron microscopy pointed out that the overall shape,the domain organization, and the way in which the Rand â subunits assemble are very similar between Rvâ3and R5â1.13,15 Fibronectin, which is the physiologicalligand of R5â1, was found to bind its receptor throughthe RGD-containing module 10 (Fn10) in a mannersimilar to cyclo(-RGDf[NMe]V-) in the Rvâ3 crystalcomplex.13 These findings, the high sequence similaritybetween R5â1 and Rvâ3 receptors (Rv:R5 53% identity;â3:â1 55% identity in the integrin’s headgroup), and theneed to get more information about the R5â1 selectivityrequirement prompted us to build a 3D model of theR5â1 receptor. A multiple sequence alignment wasperformed utilizing evolutionary information of all R andâ subunits in different organisms. The crystal structureof Rvâ3 in the bound conformation was used as atemplate, obtaining 10 3D models of R5â1 integrin bymeans of comparative protein modeling methods. Themodels, which differ mostly in the side chain orientation,were used one at a time for ligand docking studies usingthe Autodock program (see Supporting Information).Compound 1 (SJ749) was chosen as ligand because ofits potency, receptor selectivity, conformational restric-tions, and the recently published SARs (Table 1).16

Docking results obtained for the 10 receptor modelswere carefully inspected to evaluate the agreement withthe experimental data (mutagenesis, cross-linking, SARs)and the convergence and the binding free energyachieved for each simulation. SJ749 was found to fit inone R5â1 model preferentially over the others. Such acomplex was energetically minimized using 3000 stepsof steepest descent algorithm with the CVFF force field,permitting only the ligand and the side chain atoms ofthe protein within a radius of 5 Å around the ligand torelax. The stereochemical quality of the resulting modelwas checked with the program PROCHECK. The ma-jority of the residues occupied the most favored regions(81.7%) of the Ramachandran plot. Other residues

* To whom correspondence should be addressed. Phone: +49-89-289 13300. Fax: +49-89-289 13210. E-mail: kessler@ch.tum.de.

‡ Universita “Federico II” di Napoli.† Technische Universitat Munchen.

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10.1021/jm040224i CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 06/01/2005

occupied additional allowed regions (14.9%) or gener-ously allowed regions (2.5%). Only the 0.3% were indisallowed regions. The obtained complex is shown inFigure 1. Compound 1 binds in the RGD binding siteat the interface between the â-propeller of the R5subunit (blue) and the âA domain of the â1 subunit(red). The 2-aminopyridin moiety of 1 inserts into agroove at the top of the â-propeller formed primarilyby the D3-A3 and D4-A4 loops, while the carboxylategroup of the ligand coordinates the metal ion at themetal-ion-dependent adhesion site (MIDAS). The con-tact of the 2-aminopyridin moiety with the D4-A4 loopresidue (R5)-Asp227, which corresponds to (â3)-Asp218in Rvâ3,17 and the coordination of the metal ion, whichis stabilized by a hydrogen bond to the (â1)-Asn218backbone, were also observed for Rvâ3/Rvâ5 dual ligandsusing similar techniques.18 This is not surprising be-cause of the high conservation of residues in the RGDbinding site between the Rvâ3, Rvâ5, and R5â1 integrinsand is in accordance with mutagenesis data,19 cross-linking studies,20 and X-ray structure,13 which indicate

the metal ion at the MIDAS and the (â3)-Asp218 of loopD4-A4 as key elements in the interaction with RGD-like ligands.

A closer look at the different activities of SJ749analogues (2-6 in Table 1) toward the R5â1 and Rvâ3receptors indicates the importance of a carbamatefunction for the interaction with both integrins. Re-placement of the benzyl moiety with a methyl group(compounds 2 and 4) has no influence on binding of R5â1and Rvâ3 integrins, thus excluding any relevant con-tribution of such a group to the binding. Otherwise, theabsence of the carbamate function (3) leads to a signifi-cant decrease of activity, especially regarding the R5â1binding. In our model of 1 in complex with R5â1, bothcarbamate oxygens are involved in receptor bindingthrough contacts with (R5)-Ser224 and (â1)-Ser221residues within the D4-A4 and C′-R3 loops, respec-tively (Figure 1). Considering the strength of thehydrogen bond acceptor groups, the carbonyl oxygeninteraction is probably more significant than that of thesp3 oxygen.21 A comparing analysis of the R5â1 and Rvâ3receptor sequences shows that the above-mentionedserine residues are replaced by two Ala ((Rv)-Ala215 and(â3)-Ala218) in the Rvâ3 receptor (Figures 1 and 2).Nonetheless, the carbamate function seems to play arole in Rvâ3 ligand binding as well. Docking of 1 in theX-ray structure of Rvâ313 reveals that the Cbz groupcould interact with the (â3)-Lys253 side chain, whichis not present in the â1 subunit.

In our model the spirocyclic scaffold lies at theinterface between the R and â subunits, allowing aproper orientation of the pharmacophoric groups. Ac-

Table 1

compd X R1 R5â1d IC50 (nM) Rvâ3e IC50 (nM) RIIbâ3f IC50 (nM)

1 (SJ749) 2-pyridyl NHCH2a Cbz 0.18 49 >100 000

2 2-imidazolyl NHCH2b Cbz 18 41 34 000

3 2-imidazolyl NHCH2b H 230 180 1400

4 2-imidazolyl NHCH2b CO2Me 21 57 7300

5 2-imidazolyl NHCH2b CO2nBu 14 130 11 000

6 2-imidazolyl NHCH2b CO2CH2(3-pyr) 39 32 5600

7 2-dihydroimidazolyl NHCH2 Cbz 13 57 73008 2-dihydroimidazolyl NHCH2

c Cbz 1200 >1000 92009 2-benzimidazolyl NHCH2 CO2Me 1.4 34 >100 00010 2-benzimidazolyl NHCH2 CO2nBu 0.39 87 >100 000a (S,S,S)-isomer. b Mix of diastereomers at C7 (spiro). c (S,R,S)-isomer. d The IC50 values were obtained using an ELISA assay (see ref

16). e The IC50 values were obtained using a functional Rvâ3 antagonism assay involving adhesion of 293 b3-transfected cells to fibrinogen(see ref 16). f The functional assay was performed in human platelet-rich plasma (GPIIb/IIIa hPRP) (see ref 16).

Figure 1. Three-dimensional representation of the complexbetween the R5â1 receptor and 1. The R subunit is representedby blue ribbon diagram, while â subunit is in red. In bothsubunits, important side chains are shown and are in red andblue. The metal ion in the MIDAS region is represented as amagenta sphere. The Rvâ3 was superposed on R5â1 consider-ing all the backbone atoms, with side chains carbons in yellow.Mutated residues in the binding pocket are labeled with theone-letter amino acid code. The letters and the number inparentheses refer to the Rvâ3 receptor.

Figure 2. Sequence alignment of ligand-contacting loops.Mutated residues in the binding pocket are highlighted in gray.

Letters Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4205

cordingly, its absolute stereochemistry strongly influ-ences the activity toward all three integrins, which isdemonstrated by 7 and 8. Regarding the guanidinemimetic group, Smallheer et al. reported that in contrastto the Rvâ3 ligands less basic groups resulted in morepotent R5â1 antagonists.16 This could be explained bya lower acidity in the D3-A3 loop of R5â1 integrincaused by replacement of three (Rv)-Asp residues by aTrp, Phe, and Ala residue in the R5 subunit (see Figures1 and 2). Of special importance might be the mutationof Asp150 to an Ala residue in the R5-subunit: the Rvâ3crystal structure clearly indicates a double-sided coor-dination of the basic guanidine group by two Aspresidues, which is impossible in the R5 binding pocket.Moreover the (R5)-groove, in which the basic ligandgroup inserts, has a slightly different shape comparedto that of Rvâ3 because of the above-mentioned substi-tution of (Rv)-Asp150 with an Ala in the R5-subunit anddue to the replacement of (Rv)-Thr212 by (R5)-Gln221in the D4-A4 loop (Figures 1 and 2). Replacement ofthe basic group in 4 and 5 with a benzimidazole ringdoes not significantly affect the Rvâ3 potency (9 and 10),while it improves the R5â1 receptor binding (see Table1). Docking of 10 in the R5â1 integrin revealed theexpected electrostatic interactions between the carboxy-late group and the metal ion at the MIDAS region andthe bidentate salt bridge to the D4-A4 loop residue (R5)-Asp227 (Figure 3). The benzimidazole ring, which wasalready used as guanidine mimic in other integrinantagonists,22 was found to form a T-shaped interactionwith (R5)-Phe187, which is also present in the Rvâ3integrin ((Rv)-Tyr178). The proximity between the (R5)-Gln221 (Thr212 in Rv) and the benzimidazole ringallows an amide-aromatic interaction between thecarboxamide group of (R5)-Gln221 and the benzimida-zole moiety of the ligand. It is worth noting thatreplacement of (Rv)-Asp150 by (R5)-Ala159 increases thelipophilicity of this pocket. Consequently, the higherhydrophobicity of the benzimidazole in comparison tothe imidazole moiety could be the reason for theenhanced R5â1 receptor affinity. The n-butyloxycarbonylgroup inserts between the R5 and â1 subunits; as foundfor SJ749, carbamate oxygens are involved in receptorbinding through contacts with (R5)-Ser224 and (â1)-Ser221 residues. The n-butyl chain inserts in a pocketformed mainly by residues (R5)-Ile225, (R5)-Asp228,(â1)-Phe315, (â1)-Pro222, (â1)-Gly255, (â1)-Ala254 andcan be involved in weak hydrophobic interactions withthe last two residues.

In the Rvâ3 receptor, the R-N-sulfonyldiaminopro-panoic acid subunit may interact with (â3)-Arg214. Inthe R5â1 integrin, such an interaction would not beconserved because of the substitution of (â3)-Arg214 by(â1)-Gly217 and of (â3)-Arg216 by (â1)-Leu219 (Figures1 and 2). The superposition of the Rvâ3 and R5â1Connolly surfaces (Figure 4) shows that because of theabove cited mutations, a new space opened up adjacentto the RGD binding site, which is absent in Rvâ3receptor. This new pocket is formed mainly by the C′-R3 loop residues (â1)-Gly217, (â1)-Asn218, (â1)-Leu219and by the SDL residues (â1)-Ile170, (â1)-Ser171, (â1)-Thr189, and (â1)-Pro190. The roof of the pocket com-prises the SDL highly conserved sequence NPC ((â3)-Asn175, (â3)-Pro176, (â3)-Cys177)). These findingssuggest that a way to achieve Rvâ3/R5â1 selectivity isthe insertion of properly oriented bulky moieties in theligand, which should occupy the R5â1 pocket and shouldnot be tolerated by the Rvâ3 receptor. The presence ofa serine residue within the pocket would offer theopportunity to insert hydrogen bond acceptor groups onthe bulky moieties. To our knowledge, no ligand existsso far targeting this pocket.

It is worth noting that most of the R5â1 antagonistspossess two branches that mimic the Arg and the Aspresidues within the RGD recognition motif and lack ofappreciable selectivity.11 This is not surprising becausethey are designed to mimic the RGD tripeptide se-quence, which is well-known to be recognized by severalintegrins. On the other hand, we were able to achieveselectivity between Rvâ3 and RIIbâ3 by conformationalcontrol of the RGD sequence in the ligand.23a-c Weassumed that selectivity for Rvâ3 and RIIbâ3 wascaused by changing the distance between the basic andthe acid moieties of the peptidomimetics. Our hypothesishas been recently confirmed by the X-ray crystal struc-tures of RIIbâ3 in complex with different ligands.23d Inthe case of Rvâ3/R5â1 selectivity, this approach was notsuccessful, so other strategies must be explored. Withinthe R5â1 binding pocket other regions different fromthat binding Arg and Asp of the RGD sequence have tobe targeted. Compounds 1 and 10 are the most R5â1-selective ligands known to date. Both of them possessan additional chain, represented by a carbamate group,that inserts between strategically located loops, each of

Figure 3. Binding mode of 10 within the R5â1 receptor.Figure 4. Superposition of R5â1 and Rvâ3 receptors repre-sented as Connolly surfaces (gray and transparent red,respectively), with 1 docked into the R5â1 receptor. The threeâ3 residues, whose mutation in â1 opens a new space adjacentto the RGD binding site, are in white and are visible underthe red transparent Rvâ3 surface as yellow sticks.

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which bears a serine residue (Figures 1 and 2). Thus,(R5)-Ser224 and (â1)-Ser221 offer accessible attachmentpoints for hydrogen bond acceptors. Furthermore, thepresence of (R5)-Gln221 and the reduced basicity of theD3-A3 loop open new options for variation of theguanidine mimetic group. The wide pocket along thedimer border, which consists of some R5â1 peculiaramino acids, may be a special feature of such integrin,and it is worth being targeted in the effort to achievepotency and selectivity. As a result of this study, weidentified two potentially important regions in the R5â1receptor that are unique features of this integrin andmight be worth considering for drug targeting.

Supporting Information Available: Experimental Sec-tion containing details concerning the molecular modeling anddocking procedures. This material is available free of chargevia the Internet at http://pubs.acs.org.

References(1) Hynes R. O. Integrins: bidirectional, allosteric signaling ma-

chines. Cell 2002, 110, 673-687.(2) Mousa, S. A. Anti-integrin as novel drug-discovery targets:

potential therapeutic and diagnostic implications. Curr. Opin.Chem. Biol. 2002, 6, 534-541.

(3) Pancioli, A. M.; Brott, T. G. Therapeutic potential of plateletglycoprotein IIb/IIIa receptor antagonists in acute ischaemicstroke: scientific rationale and available evidence. CNS Drugs2004, 18, 981-988.

(4) (a) Brower, V. Tumor angiogenesissnew drugs on the block. Nat.Biotechnol. 1999, 17, 963-968. (b) Arndt, T.; Arndt, U.; Reuning,U.; Kessler, H. Integrins in Angiogenesis: Implications forTumor Therap. In Cancer Therapy: Molecular Targets inTumor-Host Interactions; Weber, G. F., Ed., in press.

(5) Brooks, P. C.; Clark, R. A.; Cheresh, D. A. Requirement ofvascular integrin Rvâ3 for angiogenesis. Science 1994, 264, 569-571.

(6) Shimaoka, M.; Springer, T. A. Therapeutic antagonists andconformational regulation of integrin function. Nat. Rev. 2003,2, 703-716.

(7) Burke, P. A.; DeNardo, S. J.; Miers, L. A.; Lamborn, K. R.;Matzku, S.; DeNardo, G. L. Cilengitide targeting of alpha(v)-beta(3) integrin receptor synergizes with radioimmunotherapyto increase efficacy and apoptosis in breast cancer xenografts.Cancer Res. 2002, 62, 4263-4272.

(8) Reynolds, L. E.; Wyder, L.; Lively, J. C.; Taverna, D.; Robinson,S. D.; Huang, X.; Sheppard, D.; Hynes, R. O.; Hodivala-Dikle,K. M. Enhanced pathological angiogenesis in mice lacking beta3integrin or beta3 and beta5 integrins. Nat. Med. 2002, 8, 27-34.

(9) Hynes, R. O. A reevaluation of integrins as regulators ofangiogenesis. Nat. Med. 2002, 8, 918-921.

(10) George, E. L.; Georges-Labouesse, E. N.; Patel-King, R. S.;Rayburn, H.; Hynes, R. O. Defect in mesoderm, neural tube andvascular development in mouse embryos lacking fibronectin.Development 1993, 119, 1079-1091.

(11) (a) Kumar, C. C.; Malkowski, M.; Yin, Z.; Tanghetti, E.; Yaremko,B.; Nechuta, T.; Varner, J.; Liu, M.; Smith, E. M.; Neustadt, B.;Presta, M.; Armstrong, L. Inhibition of angiogenesis and tumorgrowth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5integrin receptor antagonist. Cancer Res. 2001, 61, 2232-2238.(b) Cheng, S. PCT Int. Appl. WO 9528426 A2 19951026, 1995.Ruoslahti, E. PCT Int. Appl. WO 9514714 A1 19950601, 1995.Cleary, P. P. PCT Int. Appl. WO 9856408 A2 19981217, 1998.Labrecque, D. PCT Int. Appl. WO 2000075129 A1 20001214,2000. Ishikawa, M. PCT Int. Appl. WO 9952872 A1 19991021,1999.

(12) D’Souza, S. E.; Ginsberg, M. H.; Plow, E. F. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem. Sci.1991, 16, 246-250.

(13) Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.;Goodman, S. L.; Arnaout, M. A. Crystal structure of theextracellular segment of integrin alphaV beta3 in complex withan Arg-Gly-Asp ligand. Science 2002, 296, 151-155.

(14) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.; Holze-mann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. N-Methylatedcyclic RGD peptides as highly active and selective alpha(V)beta-(3) integrin antagonists. J. Med. Chem. 1999, 42, 3033-3040.

(15) (a) Mould, A. P.; Symonds, E. J. H.; Buckley, P. A.; Grossmann,J. G.; McEwan, P. A.; Barton, S. J.; Askari, J. A.; Craig, S. E.;Bella, J.; Humphries, M. J. Structure of an integrin-ligand

complex deduced from solution X-ray scattering and site-directedmutagenesis. J. Biol. Chem. 2003, 278, 39993-39999. (b) Takagi,J.; Strokovich, K.; Springer, T. A.; Walz, T. Structure of integrinalpha5beta1 in complex with fibronectin. EMBO J. 2003, 22,4607-4615.

(16) Smallheer, J. M.; Weigelt, C. A.; Woerner, F. J.; Wells, J. S.;Daneker, W. F.; Mousa, S. A.; Wexler, R. R.; Jadhav, P. K.Synthesis and biological evaluation of nonpeptide integrinantagonists containing spirocyclic scaffolds. Bioorg. Med. Chem.Lett. 2004, 14, 383-387.

(17) Rvâ3 sequence numbering was retrieved from the PBD database.Sequence numbering of R5 and â1 subunits started from theamino acids corresponding to the first residues of Rv and â3 inthe X-ray structure. First residues of R5 and â1 subunits werenumbered as 1 and 55 according to the template numbering.

(18) (a) Gottschalk, K. E.; Kessler, H. The structures of integrins andintegrin-ligand complexes: implications for drug design andsignal transduction. Angew. Chem., Int. Ed. 2002, 41, 3767-3774. (b) Marinelli, L.; Lavecchia, A.; Gottschalk, K. E.; Novel-lino, E.; Kessler, H. Docking studies on Rvâ3 integrin ligands:pharmacophore refinement and implications for drug design. J.Med. Chem. 2003, 46, 4393-4404. (c) Marinelli, L.; Gottschalk,K. E.; Meyer, A.; Novellino, E.; Kessler, H. Human integrinRvâ5: homology modeling and ligand binding. J. Med. Chem.2004, 47, 4166-4177.

(19) Humphries, M. J.; Symonds, E. J. H.; Mould, A. P. Mappingfunctional residues onto integrin crystal structures. Curr. Opin.Struct. Biol. 2003, 13, 236-243.

(20) Yahalom, D.; Wittelsberger, A.; Mierke, D. F.; Rosenblatt, M.;Alexander, J. M.; Chorev, M. Identification of the principalbinding site for RGD-containing ligands in the R(V)â(3) inte-grin: a photoaffinity cross-linking study. Biochemistry 2002, 41,8321-8331.

(21) Lommerse, J. P. M.; Price, S. L.; Taylor, R. IsoStar: a library ofinformation about nonbonded interactions. J. Comput. Chem.1997, 18, 758-774.

(22) (a) Keenan, R. M.; Miller, W. H.; Lago, M. A.; Ali, F. E.;Bondinell, W. E.; Callahan, J. F.; et al. Benzimidazole derivativesas arginine mimetics in 1,4-benzodiazepine nonpeptide vitronec-tin receptor (alpha v beta 3) antagonists. Bioorg. Med. Chem.Lett. 1998, 8, 3165-3170. (b) Kling, A.; Backfisch, G.; Delzer,J.; Geneste, H.; Graef, C.; Holzenkamp, U.; et al. Synthesis andSAR of N-substituted dibenzazepinone derivatives as novelpotent and selective alpha(v)beta(3) antagonists. Bioorg. Med.Chem. Lett. 2002, 12, 441-446.

(23) (a) Muller, G.; Gurrath, M.; Kessler, H.; Timpl, R. Dynamicforcing, a method for evaluating activity and selectivity profilesof RGD (Arg-Gly-Asp) peptides. Angew. Chem., Int. Ed. Engl.1992, 31, 326-328. (b) Muller, G.; Gurrath, M.; Kessler, H.Pharmacophore refinement of gpIIb/IIa antagonists based oncomparitive studies of antiadhesive cyclic and acyclic RGDpeptides. J. Comput.-Aided Mol. Des. 1994, 8, 709-730. Refine-ment and implication for drug design. J. Med. Chem. 2003, 46,4393-4404. (c) Haubner, R.; Fisinger, D.; Kessler, H. Stereoi-someric peptide libraries and peptidomimetics for designingselective inhibitors of the Rvâ3 integrin for a new cancer therapy.Angew. Chem., Int. Ed. Engl. 1997, 36, 137-1389. (d) Xiao, T.;Takagi, J.; Coller, B. S.; Wang, J.-H.; Springer, T. Structuralbasis for allostery in integrins and binding to fibrinogen-mimetictherapeutics. Nature 2004, 432, 59-67.

(24) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Improving thesensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weightmatrix choice. Nucleic Acids Res. 1994, 22, 4673-4680.

(25) Sali, A.; Potterton, L.; Yuan, F.; van Vlijmen, H.; Karplus, M.Evaluation of comparative modeling by MODELLER. Protein1995, 23, 318-326.

(26) SYBYL Molecular Modeling System, version 6.8; Tripos Associ-ates: St. Louis, MO.

(27) Head, J.; Zerner, M. C. A Broyden-Fletcher-Goldfarb-Shannooptimization procedure for molecular geometries. Chem. Phys.Lett. 1985, 122, 264-274.

(28) Vinter, J. G.; Davis, A.; Saunders, M. R. Strategic approachesto drug design. 1. An integrated software framework for molec-ular modelling. J. Comput.-Aided Mol. Des. 1987, 1, 31-55.

(29) Gasteiger, J.; Marsili, M. Iterative partial equalization of orbitalelectronegativitysa rapid access to atomic charges. Tetrahedron1980, 36, 3219-3228.

(30) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,W. E.; Belew, R. K.; Olson, A. J. Automated docking using aLamarckian genetic algorithm and an empirical binding freeenergy function. J. Comput. Chem. 1998, 19, 1639-1662.

(31) Accelrys, San Diego, CA.(32) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J.

M. PROCHECK: a program to check the stereochemical qualityof protein structures. J. Appl. Crystallogr. 1993, 26, 283-291.

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