FROM MICROMOLAR TO NANOMOLAR AFFINITY - A SYSTEMATIC-APPROACH TO IDENTIFY THE BINDING-SITE OF CGRP AT THE HUMAN CALCITONIN-GENE-RELATED PEPTIDE-1 RECEPTOR

CGRP Y-0-28-37 is known as a selective CGRP(1) receptor antagonist. In order to elucidate the essential requirements for its receptor intera ction, we performed a variety of systematic approaches by modifying th e C-terminal segments CGRP Y-0-28-37 and CGRP 27-37. N-Terminal and C- terminal segments have been synthesized, as well as chimeras which com bine segments of CGRP, adrenomedullin, and amylin. Furthermore, we car ried out an Ala scan, a Phe scan, a D-amino acid scan and a Pro scan o f CGRP 27-37. Additionally, single amino acids were replaced by those with similar biophysical properties. Receptor binding studies of all a nalogs were performed at human neuroblastoma cells SK-N-MC, which sele ctively express the hCGRP(1) receptor. On the basis of the obtained re sults, we synthesized a series of ligands with multiple amino acid rep lacements in order to optimize the exchange at each position. This app roach yielded to a series of high affinity ligands, including [D-31,P- 34,F-35] CGRP 27-37 which exhibits a 100-fold increased affinity compa red to the unmodified segment. So far, this is the smallest CGRP analo g that shows affinity in the nanomolar range.