Preclinical Study Reveals Potential of Multireceptor Antagonist to Block CGRP and PACAP

Zoe Tasma, PhD

(Credit: LinkedIn)

A recent proof-of-concept study published in Headache demonstrated that a multireceptor antagonist can be generated to simultaneously block calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) activity, both implicated in migraine and pain signaling. Overall, the preclinical study suggests that blocking both CGRP- and PACAP-responsive receptor signaling with a single molecule may be feasible and could offer scope for developing more efficacious therapies targeting these receptors.¹

In this study, researchers focused on combining 2 independent peptide antagonists, CGRP_8-37 and PACAP_6-38, into a single molecule using click chemistry at specific amino acid residues (21, 34, or 38). Once successfully generated, these multireceptor antagonists were then tested for their ability to inhibit CGRP and PACAP signaling in transfected Cos7 cells and in rat spinal cord cultures. Findings showed that the linked peptides retained similar antagonist potency to their parental antagonists. Notably, CGRP_8-37 linked at position 38 of PACAP_6-38 demonstrated more potent antagonist antagonism of CGRP activity compared with CGRP_8-37 alone.

"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," lead author Zoe Tasma, PhD, postdoctoral Fellow at Otago University in Dunedin, New Zealand, and colleagues wrote.¹

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Investigators first tested 3 truncated PACAP fragments, including PACAP_6-27, PACAP_6-30, and PACAP_6-38, to determine the most effective length for antagonizing PACAP signaling. Only PACAP_6-38 consistently inhibited PACAP-38–mediated activity and was therefore selected for further experiments. Additionally, CGRP_8-37 effectively inhibited CGRP-induced cAMP accumulation at the CGRP receptor and, with lower potency, at the AMY₁ receptor, making it the candidate to link with PACAP_6-38. Additional controls confirmed that CGRP_8-37 had no activity at PAC1, whereas PACAP_6-38 showed some antagonism at the CGRP receptor.

Researchers then had 3 peptides covalently linking CGRP_8-37 and PACAP_6-38 synthesized and tested in Cos7 cells for their ability to block cAMP accumulation at CGRP- and PACAP-responsive receptors. All 3 caused rightward shifts in CGRP dose-response curves at the canonical CGRP receptor, consistent with competitive antagonism, and also inhibited CGRP responses at the AMY₁ receptor. Dissociated rat spinal cord cell cultures were then employed to evaluate the multireceptor antagonists in a more physiologically relevant setting. Consistent with earlier studies,² CGRP triggered a time- and concentration-dependent rise in cAMP, indicating the presence of functional CGRP- and PACAP-responsive receptors.

"This study generated and characterized novel multi-receptor antagonists produced by covalently linking peptide CGRP and PACAP antagonists. This resulted in single molecules that maintained their ability to block agonist-stimulated signaling at both CGRP- and PACAP-responsive receptors. However, antagonist effectiveness was dependent on the agonist present, and the cellular background used," Tasma et al noted.¹ "These differences highlight the importance of determining agonist-dependent effects and testing antagonist functionality in cells comparable to the required in vivo setting. Targeting both CGRP and PACAP receptor systems in one molecule could be therapeutically beneficial and offer efficacy for a wider subset of patients with migraine."

REFERENCES
1. Tasma Z, Siow A, Harris PWR, Brimble MA, Hay DL, Walker CS. Development and pharmacological characterization of novel multi- calcitonin gene-related peptide and pituitary adenylate cyclase-activating peptide receptor antagonists. Headache. 2025;65(7):1064-1079. doi:10.1111/head.14916
2. Takhshid MA, Poyner DR, Chabot JG, et al. Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture. Br J Pharmacol. 2006;148(4):459-468. doi:10.1038/sj.bjp.0706750