Biomimetic screening of class B G protein-coupled receptors

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.