Identification of the Key Residue of Calcitonin Gene Related Peptide (CGRP) 27-37 to Obtain Antagonists with Picomolar Affinity at the CGRP Receptor

Abstract

Calcitonin gene related peptide (CGRP) plays an important role in the CNS and in the cardiovascular system. To identify high-affinity antagonists in competitive binding studies, we identified a novel radioactive tracer, [³H-propionyl-K²⁴]-hαCGRP 8-37, which was labeled in solution by a recently developed strategy using photolabile protecting groups at reactive side chains. This tracer was shown to be as potent as commercially available 125I-tracers for the determination of agonists and to have increased sensitivity for antagonists. We applied it to investigate the predicted turn structures centered at Pro²⁹ and Pro³⁴. The substitution at positions 29 and 34 by turn-inducing amino acid mimetica showed that these turns are highly diverse. At position 29, a hydrophobic residue is preferred that constricts the secondary structure, whereas position 34 is required to stabilize the conformation of the backbone. All high-affinity analogues showed antagonistic properties with potency similar to CGRP 8-37.