Biomimetic screening of class B G protein-coupled receptors

The 41-aa peptide Corticotropin Releasing Hormone (CRH) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the Corticotropin Releasing Hormone Receptor 1 (CRH1R), a typical member of the class-B GPCRs and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRH 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 CRH C-terminus. The resulting conjugates reconstituted potent agonists and were tested in situ in a cell-based assay. Using 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 behavioural role of CRH receptors in living mice.