Biomimetic screening of CRHR ligands

Corticotropin Releasing Hormone (CRH) is a key mediator of the stress response and the HPA axis. It is involved in the neurobiology of sereval mood disorders, including depression and anxiety. In the pituitary gland, CRH binds specifically to the CRHR1 receptor, a class B GPCR superfamily member, and stimulates the release of ACTH. Using an approach mimicking the two-domain model for signal transduction by class-B GPCR, we identified new modulators of CRH signalling using high-affinity peptide-peptide conjugate libraries. As C-terminal peptide we synthesized a cyclic 13 amino-acids peptide that possesses high affinity for the CRHR1 extracellular domain. This fixed „peptide-carrier“ is conjugated to variable fragments of human-Urocortin 1 (h-Ucn1) by chemical ligation. The coupling of the complementary azide and alkyne groups by „click-chemistry“ yielded a conjugate that fully activates the receptor with nanomolar potency. The sequential truncation of this fragment at the C-terminal and N-terminal end allowed us to determine minimal sequence that is able to activate the receptor. We then performed a systematic screening of its amino-acid substitutions, thus revealing the structural features required for agonism. Finally, we synthesized a potent small peptide CRHR1 agonist that does not require an extracellular-domain interaction.