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Devigny, Christian (2012): Biomimetic screening of class B G protein-coupled receptors: design of tailored ligands for the Corticotropin-Releasing-Hormone receptor. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of complex and multifactorial psychiatric diseases such as anxiety and mood disorders. The 41-amino acid neuropeptide Corticotropin Releasing Hormone (CRH) is a major regulator of the mammalian stress response. Upon stressful stimuli, it binds to the Corticotropin Releasing Hormone Receptor 1 (CRHR1), a typical member of the class B GPCRs and a potential novel target for the therapeutic intervention in major depressive disorder. A precise understanding of the peptide-receptor interactions is an essential prerequisite towards the development of efficient CRHR1 specific antagonists. To chemically probe the molecular interaction of CRH with its cognate receptor, a high-throughput conjugation approach which mimics the natural activation mechanism for class B GPCRs was developed. Acetylene-tagged peptide libraries were synthesized and conjugated to high-affinity azide-modified carrier peptides using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstitute potent ligands and were tested in situ for modulation of the CRHR1 activity in a cell-based assay. This approach allows to (i) define the sequence motifs which are required for receptor activation or inhibition, (ii) identify the critical functional groups and investigate structure-activity-relationships, and (iii) develop novel optimized, highly potent peptide probes which are specific for the transmembrane domain of the receptor. The membrane recruitment by a high-affinity carrier peptide enhances the potency of tethered peptides and allows the initial testing of weak fragments that otherwise would be inactive. The biomimetic screening led to the discovery of transtressin, a highly modified and potent CRHR1 transmembrane domain-specific optimized agonist (EC50 = 4 nM). Beyond its intrinsic agonistic activity, transtressin is an essential tool for the pharmacological characterization of CRHR1 antagonists in competition assays.