Abstract:
Adrenomedullin (AM) is peptide hormone which is an avid promoter of angiogenesis in a variety of models and is upregulated by hypoxia through a HIF-1a response element on the AM gene. AM administration to adult mice increases the number of blood and lymphatic vessels at the site of an injury. AM can act upon endothelial cells derived from both blood and lymphatic vessels to promote neovascularisation, proliferation and migration. Consequently, there is considerable interest in modulating this important angiogenic pathway. Although a truncated peptide antagonist (AM22-52) is reported to reduce tumour growth and vascularization, currently, there are no small molecules known to interact with AM receptors. Antagonists of AM signalling would have potential as anti-angiogenesis agents for cancer therapy, while agonists would have applications in wound healing and lymphedema settings. Many effects of AM are mediated through the AM1 receptor, which consists of two subunits: the calcitonin receptor-like receptor (CLR) (a G protein-coupled receptor) and receptor activity-modifying protein (RAMP2). A closely related peptide, calcitonin gene-related peptide (CGRP), has a similar receptor; the CGRP receptor, which is composed of CLR with RAMP1. Extensive drug discovery efforts have produced a family of CGRP receptor antagonists (e.g., telcagepant, olcegepant) with clinical activity against migraine. X-ray crystal structures of the RAMP1/CLR extra-cellular domain (ECD) complex showed that these antagonists occupy a binding site at the CLR and RAMP1 interface, and so block CGRP binding. A recent crystal structure of the AM1 receptor ECD complex with an AM peptide fragment bound revealed a distinct ligand binding groove that provides new insights for the discovery of AM1 receptor selective molecules. We have used these crystal structures to design small molecules that anchor to backbone residues in the CLR subunit (THR122) and extend into the binding groove of the AM1 receptor, mimicking the interactions of the antagonist telcagepant at the CGRP receptor. A benzimidazolone motif, known to bind THR122 of the CLR subunit, was linked to a variety of units in an effort to impart selectivity towards the AM1 receptor. Activity at the AM1 and CGRP receptors was evaluated using a combination of cAMP and β-arrestin recruitment assays. All compounds were independently screened (at least n=2) in the absence and presence of AM or CGRP to check for modulator activity or peptide-independent activity. We have identified a series of AM1 receptor selective small molecules which appear to act as positive allosteric modulators of AM activity, rather than antagonists. We report our exploration of this class of AM1 modulators, which represent first-in-class molecules.