Development of CGRP receptor peptide antagonists as potential therapeutics

Abstract

Calcitonin gene-related peptide (CGRP) is a 37 amino-acid neuropeptide that initiates intracellular signalling through binding and activation of a group of calcitonin-family class B G protein-coupled receptors (GPCR). The canonical receptor for CGRP is the heteromeric CGRP receptor, comprised of the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1). CGRP is primarily expressed in nociceptive sensory neurons that innervate peripheral tissues. CGRP displays pleiotropic biological effects that include vasodilation, inflammation, pain transmission and energy homeostasis. As CGRP activity is implicated in a variety of dysregulated conditions, the development of therapeutics that modulate or block CGRP action is an active area of research. The first developed small molecule antagonists such as olcegepant and telcagepant displayed nanomolar affinity but were discontinued due to poor oral bioavailability and liver toxicity issues respectively. However, this set the stage for the development of more successful CGRP inhibitors such as the monoclonal antibody -ubrogepant. At present, several humanised monoclonal antibodies such as erenumab and fremanezumab have shown clinical efficacy for migraine treatment and are approved by the FDA as therapeutics. Another potential therapeutic strategy is to develop peptides to block CGRP action. However, peptides typically have short half-life in vivo, which limits their utility as therapeutics for clinical use. To counteract this drawback, modifications such as lipidation can be employed to extend peptide half-life. This thesis focuses on CGRP8-37, which is a truncated form of CGRP lacking the first seven residues. CGRP8-37 is a competitive antagonist at both CGRP and Amylin 1 (AMY1) receptors, which is also responsive to CGRP. The aim was to investigate the pharmacology and biological action of lipidated analogues based on CGRP8-37 to ascertain their therapeutic potential as peptide-based antagonists of CGRP action. Chapter 3 investigates the antagonist activities of several cysteine-substituted and palmitoylated analogues based on CGRP8-37 or CGRP7-37 across several calcitonin-family receptors expressed in transiently transfected Cos-7 cells. Chapter 4 describes further characterisation of the analogues’ antagonist activities at CGRP and AMY1 receptors as well as the CGRP receptor endogenously expressed in SK-N-MC cells. Chapter 5 describes the establishment and validation of a Laser doppler technique to investigate vasodilatory action by endogenous CGRP release in vivo. Selected lipidated peptide antagonists were investigated for their ability to attenuate this response and therefore discern its CGRP antagonist activity in vivo. Findings show that palmitoylation improved antagonist activity across both CGRP and AMY1 receptors, as well as several other calcitonin family receptors. The antagonist activity of the lipidated peptides exhibited hemi-equilibrium characteristics suggesting altered binding kinetics. The lipidated peptide analogues, V8C-palmitate and K24C-palmitate, were tested in vivo and showed inhibition of CGRP action as measured through suppression of capsaicin-induced dermal vasodilatory responses in mice. Overall, these findings support lipidation as a viable option to develop CGRP peptide antagonists as potential therapeutics.