Picomolar Affinity Antagonist and Sustained Signaling Agonist Peptide Ligands for the Adrenomedullin and Calcitonin Gene-Related Peptide Receptors.
Journal
ACS pharmacology & translational science
ISSN: 2575-9108
Titre abrégé: ACS Pharmacol Transl Sci
Pays: United States
ID NLM: 101721411
Informations de publication
Date de publication:
14 Aug 2020
14 Aug 2020
Historique:
received:
30
03
2020
entrez:
25
8
2020
pubmed:
25
8
2020
medline:
25
8
2020
Statut:
epublish
Résumé
The calcitonin receptor-like class B G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) functions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) form heterodimers with CLR and determine its peptide ligand selectivity through an unresolved mechanism. The CGRP (RAMP1:CLR) and AM (RAMP2/3:CLR) receptors are proven or promising drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. Here, we used synthetic peptide combinatorial library and rational design approaches to probe the ligand selectivity determinants and develop truncated AM and CGRP antagonist variants with receptor extracellular domain binding affinities that were enhanced ∼1000-fold into the low nanomolar range. Receptor binding studies and a high-resolution crystal structure of a novel library-identified AM variant bound to the RAMP2-CLR extracellular domain complex explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in the ligand selectivity mechanism. In longer AM and CGRP scaffolds that also bind the CLR transmembrane domain, the variants generated picomolar affinity antagonists, one with an estimated 12.5 h CGRP receptor residence time, and sustained signaling agonists "ss-AM" and "ss-CGRP" that exhibited persistent cAMP signaling after ligand washout. Sustained signaling was demonstrated in primary human umbilical vein endothelial cells and the SK-N-MC cell line, which endogenously express AM and CGRP receptors, respectively. This work clarifies the RAMP-modulated CLR ligand selectivity mechanism and provides AM and CGRP variants that are valuable pharmacological tools and may have potential as long-acting therapeutics.
Identifiants
pubmed: 32832875
doi: 10.1021/acsptsci.0c00031
pmc: PMC7432658
doi:
Types de publication
Journal Article
Langues
eng
Pagination
759-772Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM103640
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM104251
Pays : United States
Informations de copyright
Copyright © 2020 American Chemical Society.
Déclaration de conflit d'intérêts
The authors declare the following competing financial interest(s): A.A.P. is the inventor on a patent for AM and CGRP variants described herein.
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