Role of conformational constraints of position 7 of the disulphide bridge of h-alpha-CGRP derivatives in their agonist versus antagonist properties.

Previous structure-activity studies have shown that the disulphide bridge of calcitonin gene-related peptide (CGRP) is important for the highly potent, CGRP receptor-mediated effects of this peptide. In this study penicillamine (Pen) was substituted for one or both of the cysteinyl residues to determine conformational and topographical properties of the disulphide bridge favourable for binding to CGRP receptors and/or receptor activation. Pen constrains the conformational flexibility of disulphide bridges in other peptides. Binding affinities were measured using a radioligand binding assay with membranes prepared from pig coronary arteries and 125I-h-alpha-CGRP. Functional effects were characterized using a previously reported pig coronary artery relaxation bioassay. The binding affinity of [Pen2]h-alpha-CGRP was not significantly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen2]h-alpha-CGRP also caused relaxation of coronary arteries. The remaining analogues either caused relaxation with significantly reduced potency or failed to relax the arteries at concentrations up to 1 x 10(-5)M. All analogues that did not relax coronary arteries contained a D-Pen in position 7 and inhibited CGRP-induced relaxation. [D-Pen2,7]h-alpha-CGRP was the most potent antagonist with a K8 value of 630 nM. This affinity is similar to that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (KBs 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effects on ligand binding and activation of CGRP receptors. The contribution of position 7 to the conformation and topography of the disulphide bridge of h-alpha-CGRP is crucial to the future design of agonists of CGRP receptors. Furthermore, position 7 is important for the development of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.