Development and pharmacological characterization of novel multi- calcitonin gene-related peptide and pituitary adenylate cyclase-activating peptide receptor antagonists.

Objective: This study aimed to provide proof-of-concept that multi-receptor antagonist peptides can be generated by covalently linking independent antagonist peptides that block calcitonin gene-related peptide (CGRP_8-37) or pituitary adenylate cyclase-activating peptide (PACAP)/vasoactive intestinal peptide (VIP) (PACAP_6-38) activity.

Background: The neuropeptides CGRP and PACAP are implicated in migraine and pain pathogenesis. CGRP and PACAP are elevated during a migraine attack, and following infusion of either peptide, patients develop migraine-like attacks. This indicates that targeting both these systems may provide therapeutic benefits. Mechanistic studies suggest that these peptides largely act independently from one another. Therefore, blocking the activity of both CGRP and PACAP simultaneously could provide a clinical advantage over individual blockade. One strategy is to develop a single antagonist capable of inhibiting the signaling of both CGRP and PACAP receptors, a multi-receptor antagonist. N-terminal truncation of CGRP and PACAP generates the antagonists CGRP_8-37 and PACAP_6-38, respectively. These are commonly used as research tools for the CGRP and PACAP receptors. These peptide antagonists were, therefore, used as the basis for the design of multi-receptor antagonists against the CGRP and PACAP receptors and to test their functionality in vitro.

Methods: To generate multi-receptor antagonists, CGRP_8-37 was linked through 1,3-dipolar cycloaddition using click chemistry to PACAP_6-38 at amino acid residues 21, 34, or 38. The ability of these multi-receptor antagonists to block CGRP activity (CGRP and AMY₁ receptors) and PACAP-38, PACAP-27, and VIP activity (PAC₁, VPAC₁, and VPAC₂ receptors) was then characterized in transfected Cos7 cells. The peptides were then further examined in pain-relevant rat spinal cord cultures, as a model of endogenous receptors.

Results: Multi-receptor antagonists were successfully generated, displaying similar antagonist potency to their parental antagonists in both transfected Cos7 cells and in spinal cord cultures. Interestingly, CGRP_8-37 linked to position 38 of PACAP_6-38 was a more potent antagonist of CGRP activity than CGRP_8-37.

Conclusion: This study provides proof-of-concept evidence for the development of potent multi-receptor antagonists capable of blocking both CGRP and PACAP activity.