The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V(32), and F(37)) adopting consistent interspatial positions with respect to one another. Peptides in the P(34) series were comparatively more flexible at the C-terminus, although a large proportion of the [D(31),P(34),F(35)]CGRP(27-37)-NH(2) calculated conformers contained a gamma-turn centered on P(34). These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27-37)-NH(2) analogues may help to appropriately orient critical residues (T(30), V(32), and F(37)) for hCGRP(1) receptor binding.
2001. Vol. 40, no 28, 8317-8325 p.