title: 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 (CRF(1)R), 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 CRF(1) 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 (EC(50) = 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.
authors.affiliation: Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Devigny
authors.forename: Francisco
authors.initials: F
authors.lastname: Perez-Balderas
authors.forename: Bastiaan
authors.initials: B
authors.lastname: Hoogeland
authors.forename: Serena
authors.initials: S
authors.lastname: Cuboni
authors.forename: Rudolf
authors.initials: R
authors.lastname: Wachtel
authors.forename: Christoph P
authors.initials: CP
authors.lastname: Mauch
authors.forename: Katharine J
authors.initials: KJ
authors.lastname: Webb
authors.forename: Jan M
authors.initials: JM
authors.lastname: Deussing
authors.forename: Felix
authors.initials: F
authors.lastname: Hausch
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D032701:Biomimetics / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D057930:Click Chemistry; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D008024:Ligands; D051379:Mice; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D054832:Urocortins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000099052:CRF Receptor, Type 1
chemical_list: D008024:Ligands; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone