Development and pharmacological characterisation of bifunctional CGRP-PACAP receptor antagonists in transfected cells and spinal cord cultures

Abstract text (incl. figure legends and references)

Question: The neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) are both implicated in migraine. Blocking the activity of these peptides simultaneously may provide a clinical advantage over individual blockade. One strategy is to develop a bifunctional ligand, capable of antagonizing both systems at once. As a starting point we utilized the known antagonism imparted by CGRP and PACAP peptide fragments, exploring different lengths of PACAP. From this, we selected CGRP_8-37 and PACAP_6-38 to attach together and assessed these molecules as bifunctional antagonists.

Methods: Peptides were synthesized in-house and CGRP_8-37 was linked to PACAP_6-38 using 1,3-dipolar cycloaddition at amino acid positions 21, 34 and 38. The potency of these peptides as bifunctional antagonists was then tested, and compared to the parent fragments. We tested antagonism against CGRP at the human CGRP and AMY₁ receptors and against PACAP-27, PACAP-38 and VIP at the human PAC₁, VPAC₁ and VPAC₂ receptors in Cos7 cells (cAMP production). Translational relevance was assessed by measuring antagonism of agonist-stimulated cAMP production in primary rat spinal cord cultures.

Results: The bifunctional antagonists generally displayed similar antagonist activity to CGRP_8-37 and PACAP_6-38 in receptor transfected Cos7 cells and spinal cord cultures. Interestingly, linking CGRP_8-37 to position 38 of PACAP_6-38 generated a peptide with greater antagonist potency than CGRP_8-37 at CGRP and AMY₁ receptors in Cos7 cells.

Conclusions: This study provides proof-of-concept that bifunctional antagonists capable of blocking both CGRP and PACAP activity can be generated.