New Class of Corticotropin-Releasing Factor (CRF) Antagonists:  Small Peptides Having High Binding Affinity for CRF Receptor

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF1R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF1 receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure–activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC50 = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

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“…and CRH 2 receptors (Grigoriadis 2005;Yamada et al 2004). X = cyclohexyl alanine; f = Dphenylalanine; EAEK = lactam bridge.…”

Section: Both Crh and Ucn Have High Affinities For The Crh-binding Prmentioning

confidence: 99%

Biomimetic Screening of Class-B G Protein-Coupled Receptors

et al. 2011

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“…They are antagonists because they occupy the major binding site, thus blocking agonist binding, but they cannot induce receptor signaling because the first eight residues are absent. Such peptide antagonists are astressin (42) and the short CRF-based analogs comprising residues 30-41 (9), which bind to the ECD1 of CRF-R1 in a similar conformation as astressin (Fig. 1B and SI Table 3) (33).…”

Section: Crf Ligand-receptor Interactions Have a Common Binding Modementioning

confidence: 99%

“…These belong to the peptide hormone B1 family (family B1 GPCRs), comprising receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, and parathyroid hormone. Two CRF receptors, CRF-R1 and CRF-R2, have been cloned in mammals (6, 7).Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was also observed for the receptors (11,12).…”

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confidence: 99%

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Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

Grace¹,

Perrin

Gulyás

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

126

175

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The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2␤ in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.3D structure ͉ astressin ͉ corticotropin releasing factor ͉ NMR T he ability of the body to adapt to stressful stimuli and the role of stress maladaptation in human diseases has been intensively investigated. Corticotropin releasing factor (CRF) (1), a 41-residue peptide, and its three paralogous peptides, urocortin (Ucn) 1, 2, and 3, play important and diverse roles in coordinating endocrine, autonomic, metabolic, and behavioral responses to stress (2, 3). CRF family peptides and their receptors are also implicated in the modulation of additional central nervous system functions including appetite, addiction, hearing, and neurogenesis and act peripherally within the endocrine, cardiovascular, reproductive, gastrointestinal, and immune systems (4, 5). CRF and related ligands initially act by binding to their G protein-coupled receptors (GPCRs). These belong to the peptide hormone B1 family (family B1 GPCRs), comprising receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, and parathyroid hormone. Two CRF receptors, CRF-R1 and CRF-R2, have been cloned in mammals (6, 7).Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was a...

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“…Recently, Yamada et al described a twelve amino acid long peptide antagonist based on the C-terminus of CRH, indicating that this short C-terminal sequence is enough for binding the extracellular domain of the CRHR 1 (CRHR 1 -ECD1) (Yamada et al 2004).…”

Section: Peptide Antagonistsmentioning

confidence: 99%