Structure-activity relationship of human calcitonin-gene-related peptide

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each other's receptors. The present study was undertaken to examine the structure prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxy-amidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, α and β, were equipotent, and were both at least 1000-fold less potent than calcitonin; (2) in the blood flow and oedema assays, both CGRP peptides, α and β, were equipotent and were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxy-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxy-amidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotrypic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala¹-Lys³⁵): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results sugggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the α and β forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.