ROLE OF CONFORMATIONAL CONSTRAINTS OF POSITION-7 OF THE DISULFIDE BRIDGE OF H-ALPHA-CGRP DERIVATIVES IN THEIR AGONIST VERSUS ANTAGONIST PROPERTIES

Previous structure-activity studies have shown that the disulphide bri dge of calcitonin gene-related peptide (CGRP) is important for the hig hly potent, CGRP receptor-mediated effects of this peptide. In this st udy penicillamine (Pen) was substituted for one or both of the cystein yl residues to determine conformational and topographical properties o f the disulphide bridge favourable for binding to CGRP receptors and/o r receptor activation. Pen constrains the conformational flexibility o f disulphide bridges in other peptides. Binding affinities were measur ed using a radioligand binding assay with membranes prepared from pig coronary arteries and I-125-h-alpha-CGRP. Functional effects were char acterized using a previously reported pig coronary artery relaxation b ioassay. The binding affinity of [Pen(2)]h-alpha-CGRP was not signific antly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen(2)]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 inhibi ted CGRP-induced relaxation. [D-Pen(2,7)]h-alpha- CGRP was the most po tent antagonist with a K-B value of 630 nM. This affinity is similar t o that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (K-B, 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effe cts on ligand binding and activation of CGRP receptors. The contributi on 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 developme nt of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.