The aqueous solution structure of the cyclic pentapeptide cyclo(-Ser-D-Leu-Asp-Val-Pro-) has been determined by two-dimensional 1H-NMR spectroscopy, combined with a conformational search and distance-geometry calculations. As many as five conformers in slow exchange were observed, and the rate of interconversion between components was measured from the build-up rates of exchange peaks. NMR data allowed the structures of the two predominant conformers to be determined. The major component (66%) contained a cis-proline as part of a type-VIa2 beta-turn encompassing residues Asp-Val-cis-Pro-Ser. The second component (16%) contained only trans-amide bonds, and a type-VIII beta-turn formed by residues Val-Pro-Ser-D-Leu. These structures are discussed in relation to the (phi, psi), space available to the cyclic pentapeptide, determined by a conformational search, and in relation to previously published cyclic-pentapeptide structures. The molecule exhibits activity in a scintillation-proximity assay for the inhibition of the interaction between the integrin very-late antigen-4 (VLA-4; alpha 4 beta 1) and vascular-cell-adhesion molecule-1 (VCAM-1). The structure/activity relationship of the LDV sequence is discussed and related to the recently published X-ray structure of VCAM-1. The relevance of the work to the design of anti-inflammatory drugs is discussed.

1 The main aim of this study was to identify putative b-bends and the role of the N-and Cterminus in the CGRP receptor antagonist ha CGRP 8 ± 37 , which was measured against ha CGRP inhibition of twitch responses in the rat prostatic vas deferens. 2 With a bend-biasing residue (proline) at position 16 in ha CGRP 8 ± 37 (10 75 M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pK B 5.6+0.1 at 10 75 M). Proline at position 19 within ha CGRP 8 ± 37 (10 75 M) was an antagonist (apparent pK B 5.8+0.1). 3 Incorporation of a bend-forcing structure (beta-turn dipeptide or BTD) at either positions 19,20 or 33,34 in ha CGRP 8 ± 37 (10 75 M) antagonized ha CGRP responses (apparent pK B 6.0+0.1 and 6.1+0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within ha CGRP 8 ± 37 competitively antagonized responses to ha CGRP (pA 2 6.2; Schild plot slope 1.0+0.1). 4 Ha CGRP 8 ± 37 analogues (10 75 M), substituted at the N-terminus by either glycine 8 , or des-NH 2 valine 8 or proline 8 were all antagonists against ha CGRP (apparent pK B 6.1+0.1, 6.5+0.1 and 6.1+0.1, respectively), while ha CGRP 8 ± 37 (10 75 M) substituted in three places by proline 8 and glutamic acid 10,14 was inactive. 5 Replacement of the C-terminus by alanine amide 37 in ha CGRP 8 ± 37 (10 75 M) failed to antagonize ha CGRP responses. 6 Peptidase inhibitors did not alter either the agonist potency of ha CGRP or the antagonist a nities of ha CGRP 8 ± 37 BTD 19,20 and 33,34 and ha CGRP 8 ± 37 Gly 8 (against ha CGRP responses). 7 In conclusion, two b-bends at positions 18 ± 21 and 32 ± 35 are compatible with high a nity by BTD and is the ®rst approach of modelling the bioactive structure of ha CGRP 8 ± 37 . Further, the Nterminus of ha CGRP 8 ± 37 is not essential for antagonism, while the C-terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

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