Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains

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)-NH2 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)-NH2. In the study presented here, conformational and structural features in CGRP(27-37)-NH2 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)-NH2 resulted in [D-31,P-34,F-35]CGRP(30-37)-NH2, 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)-NH2 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)-NH2 peptides, [D-31,X-34,F-35]CGRP(27-37)-NH2 (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)-NH2 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)-NH2 analogues may help to appropriately orient critical residues (T-30, V-32, and F-37) for hCGRP(1) receptor binding.