Migraine is a debilitating headache disorder. The disease has neurovascular origin and migraine attacks can be elicited by vasodilative neuropeptides such as alpha calcitonin gene-related peptide (αCGRP). Antagonizing CGRP actions in migraine patients has proven clinically efficient. Here, we present a pipeline for development of a peptide-based hCGRP receptor antagonist with increased half-life capable of antagonising the vasodilatory effect of hαCGRP. A series of hαCGRP8-37 analogues carrying a C18-or C20-diacid lipidation was screened for their antagonism against the hCGRP receptor. hαCGRP8-37 analogues with a C20-diacid were 2-6 fold more potent than analogues conjugated with a C18-diacid. Half-life of hαCGRP8-37 analogues carrying a C20-diacid was estimated in mice in a pilot study (n = 1-2). Half-lives ranged from 7.3 to 13.7 h. An hαCGRP8-37 analogue conjugated with a C20 diacid at position 25 was subjected to an amino acid substitution scan to identify mutations that could further enhance hCGRP receptor antagonism. Substituting alanine with serine at position 36 resulted in a ~ 4 fold gain of potency. Vasodilative actions of hαCGRP were successfully antagonized by hαCGRP8-37 analogues carrying a C20 diacid at position 25. Our findings demonstrate that lipidation can improve hαCGRP8-37 pharmacokinetics while maintaining hαCGRP antagonism, thus demonstrating potential for a peptide-based migraine treatment strategy.
© 2024. The Author(s).

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.
© 2024 The Author(s).

Inflammation is a protective response to pathogens and injury. To be effective it needs to be resolved by endogenous mechanisms in order to avoid prolonged and excessive inflammation, which can become chronic. Specialized pro-resolving mediators (SPMs) are a group of lipids derived from omega-3 fatty acids, which can induce the resolution of inflammation. How SPMs exert their anti-inflammatory and pro-resolving effects is, however, not clear. Here, we show that SPMs such as protectins, maresins, and D-series resolvins function as biased positive allosteric modulators (PAM) of the prostaglandin E2 (PGE2) receptor EP4 through an intracellular binding site. They increase PGE2-induced Gs-mediated formation of cAMP and thereby promote anti-inflammatory signaling of EP4. In addition, SPMs endow the endogenous EP4 receptor on macrophages with the ability to couple to Gi-type G-proteins, which converts the EP4 receptor on macrophages from an anti-phagocytotic receptor to one increasing phagocytosis, a central mechanism of the pro-resolving activity of synthetic SPMs. In the absence of the EP4 receptor, SPMs lose their anti-inflammatory and pro-resolving activity in vitro and in vivo. Our findings reveal an unusual mechanism of allosteric receptor modulation by lipids and provide a mechanism by which synthetic SPMs exert pro-resolving and anti-inflammatory effects, which may facilitate approaches to treat inflammation.

Peptide-based drug discovery has surged with the development of peptide hormone-derived analogs for the treatment of diabetes and obesity. Machine learning (ML)-enabled quantitative structure-activity relationship (QSAR) approaches have shown great promise in small molecule drug discovery but have been less successful in peptide drug discovery due to limited data availability. We have developed a peptide drug discovery platform called streaMLine, enabling rigorous design, synthesis, screening, and ML-driven analysis of large peptide libraries. Using streaMLine, this study systematically explored secretin as a peptide backbone to generate potent, selective, and long-acting GLP-1R agonists with improved physicochemical properties. We synthesized and screened a total of 2688 peptides and applied ML-guided QSAR to identify multiple options for designing stable and potent GLP-1R agonists. One candidate, GUB021794, was profiled in vivo (S.C., 10 nmol/kg QD) and showed potent body weight loss in diet-induced obese mice and a half-life compatible with once-weekly dosing.

GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca2+-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca2+, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.