Structural studies of potent constrained RGD peptides - Journal of the

Robert S. McDowell, and Thomas R. Gadek ... Olga Kononova , Rustem I. Litvinov , Dmitry S. Blokhin , Vladimir V. Klochkov , John W. Weisel , Joel S. B...
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VOLUME 114, NUMBER 24 NOVEMBER 18, 1992 0 Copyright 1992 by rhe American Chemical Society

TO” OF THE AMERICAN CHEMICAL SOCIETY

Structural Studies of Potent Constrained RGD Peptides ‘Robert S. McDoweU* and Thomas R. Gadek Contribution from the Department of Bioorganic Chemistry, Genentech, Inc., 460 Point Son Bruno Boulevard, South San Francisco, Califarnia 94080. Received April 17, 1992

Abstract The threedimensionalstructure of a highly potent cyclic peptide antagonist of fibrinogezqlycoproteinIIbIIIa association was determined using NMR methods combined with molecular dynamics refinement. The molecule, which contains the Arg-Gly-Asp (RGD) recognition sequence, appears to be conformationallyrigid in water at neutral pH, displaying a well-defined geometry of the RGD sequence. By comparison, related isomers with reduced potencies are found to have less well-defined geometries under similar conditions. The structure of this molecule may therefore provide a three-dimensional template for the design of nonpeptidic RGD mimetics.

Introduction Glycoprotein IIbIIIa (GPIIbIIIa) appears on the surface of platelets as a noncovalent heterodimeric complex’ and has been shown to interact with fibrinogen,2fibronectin? vitronectin: von Willebrand factor: and thrombospondin.6 These adhesive proteins contain Arg-Gly-Asp (RGD) sequences which serve as a basic recognition feature for binding to GPIIbIIIa7 and certain other integrins.”lo In various binding studies, small peptides containing the RGD fragment have been shown to successfully compete with the larger proteins.”-14 The interaction between fibrinogen and GPIIbIIIa is the common ultimate event in the aggregation cascade regardless of the mechanism of platelet activati~n.’~We and others have therefore sought to develop an RGD-based antagonist of the GPIIbIIIa-fibrinogen interaction as a stimulusindependent inhibitor of platelet aggregation. A class of snake venom proteins which contain the RGD sequence, collectively termed disintegrins,16 are among the most potent inhibitors of GPIIbIIIa-fibrinogen binding reported. Included in this family are echi~tatin,’~ trigramin,l*and kistrin.19 These molecules are likewise potent inhibitors of platelet aggregation. Barker and co-workers have recently reported a series of thioether-bridged cyclic peptides of the general form (cydo)-S-acetyl1-X2-Arg3-Gly4-AspS-Cys6-OH, which are also effective inhibitors of GPIIbIIIa-fibrinogen binding as measured by both ELISA-format assays and platelet aggregation studies.20 By combining the chiral oxidation of the thioether with the r position X2, we used this peptide framework placement of ~ T y in To whom correspondence should be addressed.

Table I. Potencies of RGD-Based GPIIbIIIa Antagonists against

Human Platelet Aggregation

COOH

D.Tyr

-Arg -Gly -

R2

compd

Rl

1

0

2 3

e.

..

kistrin GRGDV Electron pair.

R2

IC50 (PM)

.a

0.15 2.25

. 0 ..

0.30 0.12

75.00

to generate compounds 1-3 (Table I). While the sulfide 3 is significantly less potent than the disintegrin kistrin, introduction (1) Jennings, L.K.;Phillips, D. R. J. Bid. Chem. 1982,257, 10458-10466. (2) Bennett, J. S.;Hoxie, J. A.; Leitman, S. F. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 2417-2421. ( 3 ) Ginsberg, M. H.; Fonyth, J.; Lightsey, A.; Chediak, J.; Plow, E.F.J. Clin. Invest. 1983, 71, 619-624. (4) Pytela, R.; Pierschbacher, M. D.; Ginsberg, .M. H.; Plow, E. F.; Ruoslahti, E.Science 1986, 231, 1559-1 562.

0002-786319211514-9245$03.00/00 1992 American Chemical Society

McDowell and Gadek

9246 J. Am. Chem. SOC.,Vol. 114, No. 24, 1992 Table II. Observed Chemical Shifts (ppm) Coefficients for Compound l a hydrogen Acl D-Tyr2 Arg' NH 8.93" 8.73 H" 3.98' 4.49 4.11 Hd

H" He'

2.86 3.05

H V

H6 H'

-A6/AT

'Broad

7.13' 6.82b

and Amide Temperature Gly4

AspS

Cys6

7.83 3.6SC 4.29c

8.48 4.66

8.36 4.77

2.53c 2.74c

3.24 3.50

8.0

5.5

1.35 1.80 0.9@ 2.95b 8.7

peak; poorly resolved. Diastereotopic assignment ambiguous.

4.3

Degenerate hydrogens.

of the chiral sulfoxide generated two diastereomers, 1 and 2,with vastly different potencies. Peptide 2 is much weaker than 3,while 1 is equipotent to kistrin in platelet aggregation assays, with an ICs0 of 0.150 pM. Similar trends were observed with other hydrophobic residues in the X2 position. These observations prompted us to assume that the primary role of the sulfoxide was to affect the geometry of the RGD equitope, suggesting that the solution-phase conformations of these peptides are largely responsible for their differential activities. We tested this assumption by studying the solution conformations of 1-3 in water using N M R and molecular dynamics. Peptide Synthesis. Peptides synthesized using natural abundance I5N are designated la-3a; peptides incorporating 15N-labeled cysteine are designated lb-3b. The syntheses and biological activities of peptides la-3a and GRGDV have been described previously.20 Cysteine enriched with lsN was incorporated into lb-3b using the same Boc protection schemes and polystyrene solid support employed for compounds la-3a. The purification and biological activity of kistrin, measured using an identical platelet aggregation assay, have been reported p r e v i o u ~ l y . ~ ~ NMR Measurements. Assignment of the 'H NMR Spectra of 1-3. The observed spectral dispersion enabled a facile assignment of every 'H rwnance in the 1D spectrum of compounds 1-3. The spin systems of each amino acid residue were identified in the 'H-IH COSY and the Ac', Arg3, and Glf residues were assigned on the basis of the observed chemical shifts and the characteristic scalar coupling relationships. The spin systems of the remaining three residues (bTyr2,Asps, and Cy&) were too (5) Ruggeri, 2. M.; Bader, R.; DeMarco, L. Proc. Nail. Acad. Sci. U.S.A. 1982, 79, 6038-6044. (6) Plow, E. F.; McEver, R. P.; Coller, B. S.; Woods, V. L., Jr.; Marguerie, G. A.; Ginsberg, M. H. Blood 1985,66, 724-727. (7) Ruoslahti. E.; Pierschbacher. M. D. Cell 1986. 44. 517-518. (8) v e l a , R.; Pierschbacher, M. D.; Argrave-s, S.; Suzuki, S.;Ruoslahti, E. Meihods Enzymol. 1987, 144, 475-489. (9) Hynes, R. 0. Cell 1987, 48, 549-554. (10) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491-497. ( 1 1) Plow, E. F.;Pierschbacher, M.D.; Ruoslahti, E.; Marguerie, G. A,; Ginsberg, M. H. Proc. Nail. Acad. Sci. U.S.A. 1985,82, 8057-8061. (12) Plow, E. F.; Ginsberg, M. H. Prog. Hemosiasis Thromb. 1989, 9, 117-1 56. (13) Haverstick, D. M.; Cowan, J. F.; Yamada, K. M.; Santoro, S.A. Blood 1985, 66, 946-952. (14) Gartner, T. K.; Bennett, J. S.J. Biol. Chem. 1985,260, 11891-11894. (15) Kieffer, N.; Phillips, D. R. Annu. Rev. Cell Biol. 1990, 6, 329-357. (16) Gould, R. J.; Polokoff, M. A,; Friedman, P. A,; Huang, T.-F.; Holt, J. C.; Cook, J. J.; Niewiarowski, S . Proc. SOC.Exp. Biol. Med. 1990, 195, 168-171. (17) Gan, 2.-R.; Gould, R. J.; Jacobs, J. W.; Friedman, P. A,; Polokoff, M. A. J. Biol. Chem. 1988. 263. 19827-19832. (18) Huang, T.-F.; Holt,'J. C.: Lukasiewicz, H.; Niewiarowski, S . J. Biol. Chem. 1987, 262, 16157-16163. (19) Dennis, M.S.; Henzel, W. J.; Pitti, R. M.; Lipari, M. T.; Napier, M. A.; Deisher, T. A.; Bunting, S.; Lazarus, R. A. Proc. Natl. Acad. Sci. U.S.A.

-.- . (20) Barker, P. L.; Bullens, S.; Bunting, S.; Burdick, D. J.; Chan, K. S.;

1990.87. 2471-2475 - -

Deisher, T.; Eigenbrot, C.; Gadek, T. R.; Gantzos, R.; Lipari, M. T.; Muir, C. D.; Napier, M. A.; Pitti, R. M.; Padua, A,; Quan, C.; Stanley, M.; Struble, M.; Tom, J. Y. K.; Burnier, J. P. J. Med. Chem. 1992, 35, 204C-2048. (21) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64, 2229-2246. (22) Bax, A.; Freeman, R. J. Magn. Reson. 1981, 44, 542-561.

Table III. Coupling Constants for Compounds 1-3 ' J (Hi9 residue atom pair 1 2' 3' D-Tyr2 Ha,HR2 11.7 9.7 11.8 Ha,HB' 5.9 6.3 5.9 Arg' NH, Ha 8.8 9.8 7.8 Ha,HR2 11.5 10.2 11.2 H", HB' 3.5 3.9 3.4 ~ 1 ~ 4 NH, Ha 3.8 4.9 4.9 NH, Hd 9.8 4.9 4.9 AspS NH, Ha 7.8 7.8 7.8 Ha,HR2 8.8 6.4 5.8 Ha,HB' 5.9 6.4 5.8 Cys6 NH, H" 9.8 6.8 7.8 Ha,HR2 12.7 1.3 5.6 Ha,H@' 2.9 4.4 5.6 lSN,H@