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The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.

ABSTRACT: The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

SUBMITTER: Woolley MJ

PROVIDER: S-EPMC3785825 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.

Woolley Michael J MJ Watkins Harriet A HA Taddese Bruck B Karakullukcu Z Gamze ZG Barwell James J Smith Kevin J KJ Hay Debbie L DL Poyner David R DR Reynolds Christopher A CA Conner Alex C AC

Journal of the Royal Society, Interface 20130918 88

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271-294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 resid ...[more]

PMID: 24047872

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Project description:BACKGROUND:Trans-spinal direct current stimulation (tsDCS) is a non-invasive technique with promising neuromodulatory effects on spinal cord (SC) circuitry. Computational studies are essential to guide effective tsDCS protocols for specific clinical applications. This study aims to combine modelling and experimental studies to determine the electrode montage that maximizes electric field (E-field) delivery during cervical tsDCS. METHODS:Current and E-field distributions in the cervical SC were predicted for four electrode montages in a human realistic model using computational methods. A double-blind crossover and randomized exploratory study was conducted using the montage that maximized E-field delivery. tsDCS was applied for 15 min in 10 healthy subjects (anodal, cathodal, sham, with polarity assigned to the cervical electrode), with a current intensity of 2.5 mA, resulting in a total current charge density delivery of 90 mC/cm2. Upper limb motor (transcranial magnetic stimulation) and sensory evoked potentials (MEP, SEP), M-waves, H-reflex and F-wave responses were analysed. Central and peripheral conduction times were determined using MEP. Repeated measures ANOVA and Friedman test were used for statistical analysis (significance level α = 0.05). RESULTS:All montages presented higher current density and E-field magnitudes in the cervical SC region between the electrodes. However, electrodes at C3 and T3 spinous processes (C3-T3) originated the highest E-field magnitude (0.50 V/m). Using C3-T3 montage we observed significant changes in N9 SEP latency (p = 0.006), but significance did not persist in pairwise comparisons (sham-anodal: p = 0.022; sham-cathodal: p = 0.619; anodal-cathodal: p = 0.018; α = 0.017, Bonferroni corrected). MEP latency and central motor conduction time (CMCT) modified significantly on stimulation (p = 0.007 and p = 0.015, respectively). In addition, pairwise comparisons confirmed significant differences between sham and cathodal conditions after Bonferroni correction for MEP latency (sham-anodal: p = 0.868; sham-cathodal: p = 0.011; anodal-cathodal: p = 0.023) and CMCT (sham-anodal: p = 0.929; sham-cathodal: p = 0.010; anodal-cathodal: p = 0.034). CONCLUSIONS:Computational models predicted higher E-field delivery in the cervical SC for the C3-T3 montage. Polarity-dependent effects in motor responses were reported using this montage consistent with spinal motor modulation. tsDCS experimental protocol designs should be guided by modelling studies to improve effectiveness.

Project description:Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

Project description:OBJECTIVE:Evaluate the safety and tolerability of oral rimegepant when used for acute treatment concomitantly with a monoclonal antibody (mAb) targeting the calcitonin gene-related peptide (CGRP) ligand or receptor (CGRP mAb) for the preventive treatment of migraine. BACKGROUND:The efficacy of CGRP mAbs for the preventive treatment of migraine and the small molecule CGRP receptor antagonist rimegepant for acute treatment has been demonstrated in randomized controlled clinical trials. Over the past few years, the US Food and Drug Administration has approved 4 CGRP mAbs for the preventive treatment of migraine and 2 small molecule CGRP receptor antagonists for the acute treatment of migraine. A previous case report of 2 patients receiving concomitant treatment with rimegepant and erenumab suggested that rimegepant may be safely used as acute treatment in patients who are also receiving a preventive regimen involving CGRP mAbs. We report here 13 additional patients with migraine who simultaneously used rimegepant and either erenumab, fremanezumab, or galcanezumab and assess the rate of on-treatment adverse events (AEs). METHODS:This was a substudy nested within a multicenter, open-label, long-term safety study in adults with 2-14 monthly migraine attacks of moderate to severe pain intensity. A subgroup experiencing 2-8 monthly attacks and taking a stable dose of a CGRP mAb also took rimegepant 75 mg as needed up to once daily for acute treatment for 12 weeks. RESULTS:The 13 patients (11 women [85%]; mean age 49.9 years) enrolled in the substudy were being treated with CGRP mAbs (erenumab [n = 7], fremanezumab [n = 4], or galcanezumab [n = 2]). Mean (SD) time in the rimegepant treatment period was 9.6 (4.6) weeks. Mean (SD) 4-week rimegepant exposure was 7.8 (5.5) doses; a total of 224 doses were taken. Five (38%) patients reported ?1 on-treatment AE. Of these, 2 (15%) patients had mild or moderate nasopharyngitis; no other AEs occurred in ?2 patients. Three patients had AEs of mild or moderate severity that were considered potentially treatment-related. No patients had serious AEs, AEs leading to discontinuation, or aminotransferase levels >3× the upper limit of normal. CONCLUSION:Rimegepant, when used as an oral acute treatment in patients receiving CGRP mAbs as preventive treatment, was well tolerated; no safety issues were identified. Studies involving larger patient populations are needed to confirm these findings.

Dataset Information

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.

Publications

The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach.

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