Pharmacological characterisation of cysteine-substituted calcitonin gene-related peptide antagonists in cos-7 cells

A. Jamaluddin^1,2, E. T. Williams³, D. L. Hay^1,2, M. A. Brimble^3,2, P. W. Harris^3,2, C. S. Walker^1,2, K. M. Loomes^1,2. ¹School of Biological Sciences, University of Auckland, Auckland, New Zealand, ²Maurice Wilkins Centre, Auckland, New Zealand, ³School of Chemical Sciences, University of Auckland, Auckland, New Zealand.

Introduction: Calcitonin gene-related peptide (CGRP) is a neuropeptide implicated in pain and metabolic disease (1, 2). There is currently great interest in blocking CGRP activity to treat and prevent migraine. CGRP has activity through the CGRP and Amylin₁ (AMY₁) receptors, although the role of each receptor in CGRP biology is poorly understood. CGRP_8-37 is a truncated form of the CGRP peptide that can act as an antagonist of both receptors. Previous structure-activity studies between the CGRP peptide and receptor provided useful background information to modify and develop an optimised CGRP_8-37. We have initially generated cysteine-substituted Val-8, Lys-24, and Lys-35 analogues of CGRP_8-37 as well as CGRP_7-37 to determine the effects of these substitutions to inform strategies for modifying the peptide at these positions.

Method: Peptides were synthesised using Fmoc-solid phase peptide synthesis. The analogues were characterised for efficacy and receptor specificity in vitro with cyclic-AMP assays in Cos-7 cells expressing either human CGRP or AMY₁receptors (3). The cells were stimulated with CGRP in the absence or presence of different antagonists. pK_B values were determined using the Gaddum/Schild regression equation in Graphpad Prism 7. Statistical significance was determined by unpaired t-test or one-way ANOVA with post-hoc Dunnett’s test. All experimental data were combined from a minimum of three independent repeats and presented as mean ± SEM.

Results: Unmodified CGRP_8-37 synthesised behaved as expected at both receptors. Based on the pK_B values calculated, only cysteine substitution at position 8 of CGRP_8-37 showed significant reduction in antagonism as compared to CGRP_8-37 at both CGRP and AMY₁ receptor (Table 1). Cysteine substitution at positions 24 and 35 did not affect antagonist potency.

Conclusions: Overall, the data demonstrate retention of antagonistic activity which indicates possible locations for further modifications (such as lipidation or PEGylation) to further improve the peptide’s pharmacokinetic profile.

References:

1. Hay DL and Walker CS (2017). Headache: The Journal of Head and Face Pain 7(4), 625-636.

2. Walker CS et al. (2010). Endocrinology 151(9): 4257-4269.

3. Woolley MJ et al. (2017). Biochemical Pharmacology In Press,Corrected Proof: https://doi.org/10.1016/j.bcp.2017.07.005