Biomimetic Screening of Class-B G Protein-Coupled Receptors

Abstract

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.

Myricetin: a potent approach for the treatment of type 2 diabetes as a natural class B GPCR agonist

“…Class B GPCRs are activating receptors for many endocrine peptide hormones (32, 33), including glucose‐dependent insulinotropic polypeptide, glucagon, parathyroid hormone, vasoactive intestinal peptide, secretin, corticotropin‐releasing factor, calcitonin, and GLP‐1. The endogenous ligands of class B GPCRs are typically 30–40 amino acids in size, which limits their clinical applications owing to poor stability and the requirement of injections (32), like GLP‐1 peptide.…”

“…The endogenous ligands of class B GPCRs are typically 30–40 amino acids in size, which limits their clinical applications owing to poor stability and the requirement of injections (32), like GLP‐1 peptide. Unfortunately, traditional small‐chemical molecules minimally modulate the activities of class B GPCRs because of the unique structural architectures and activation mechanisms used by these GPCRs (33). Although many efforts have been made to screen small‐class B GPCR agonists, much difficulty has been encountered in identifying small organic molecules by class B GPCRs.…”

Structure–activity relationships for α‐calcitonin gene‐related peptide

“…The mechanism by which the endogenous ligands for family B GPCRs, like CGRP, bind to and activate their receptors is of considerable interest (Neumann et al ., ; Parthier et al ., ; Watkins et al ., ) because this can lead to the design of novel antagonists and agonists. Understanding the structure–activity relationships of natural peptides has led to the development of several stable and efficacious peptide‐based agonists of family B GPCRs that have ultimately become therapeutics (Shimizu et al ., ; Devigny et al ., ; Garber, ). This review considers recent developments in the structure–activity relationship for CGRP and how these might be interpreted in the light of current knowledge of the binding of ligands to family B GPCRs.…”

The structure and function of the glucagon‐like peptide‐1 receptor and its ligands

“…The importance of the ligand's α‐helical secondary structure in the two‐domain model for mediating the initial interaction with the NTD has been reviewed (Parthier et al ., 2009), while a common helix‐capping model to explain how the various ligands of Family B GPCRs might activate their receptors has been proposed (Neumann et al ., 2008). An example of how knowledge of the two‐domain model can be used to design and synthesize novel ligands has recently been described in an elegant study by Devigny et al . (2011), who produced high potency probes with in vivo activity.…”