Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors

The calcitonin receptor-like G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) actions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) determine CLR ligand selectivity through an unresolved mechanism. CLR-RAMP complexes are drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. We used combinatorial peptide library and rational design approaches to probe selectivity determinants and develop short AM and CGRP variants with [~]1000-fold increased receptor extracellular domain affinities. Binding and structural studies explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in selectivity. In longer scaffolds that also bind the CLR transmembrane domain the variants generated picomolar affinity antagonists, one with an estimated 12.5 hr CGRP receptor residence time, and sustained signaling agonists ss-AM and ss-CGRP. This work clarifies the RAMP-modulated ligand selectivity mechanism and provides AM and CGRP variants with promise as long-acting therapeutics.