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 Y0-28-37 is known as a selective CGRP1 receptor antagonist. In order to elucidate the essential requirements for its receptor interaction, we performed a variety of systematic approaches by modifying the C-terminal segments CGRP Y0-28-37 and CGRP 27-37. N-Terminal and C-terminal segments have been synthesized, as well as chimeras which combine segments of CGRP, adrenomedullin, and amylin. Furthermore, we carried out an Ala scan, a Phe scan, a D-amino acid scan and a Pro scan of CGRP 27-37. Additionally, single amino acids were replaced by those with similar biophysical properties. Receptor binding studies of all analogs were performed at human neuroblastoma cells SK-N-MC, which selectively express the hCGRP1 receptor. On the basis of the obtained results, we synthesized a series of ligands with multiple amino acid replacements in order to optimize the exchange at each position. This approach yielded to a series of high affinity ligands, including [D31,P34,F35] CGRP 27-37 which exhibits a 100-fold increased affinity compared to the unmodified segment. So far, this is the smallest CGRP analog that shows affinity in the nanomolar range.
Rist
B
B
Department of Pharmacy, ETH Zürich, Zürich, Switzerland.
Entzeroth
M
M
Beck-Sickinger
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015740:Calcitonin Gene-Related Peptide / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008832:Microchemistry; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D009842:Oligopeptides; D010446:Peptide Fragments; D018015:Receptors, Calcitonin Gene-Related Peptide; C071243:calcitonin gene-related peptide (28-37); D015740:Calcitonin Gene-Related Peptide