Targeting the adrenomedullin-2 receptor for the discovery and development of novel anti-cancer agents.

INTRODUCTION: Adrenomedullin (AM) is a peptide responsible for many physiological processes including vascular health and hormone regulation. Dysregulation of AM signaling can stimulate cancers by promoting proliferation, angiogenesis and metastasis. Two AM receptors contribute to tumor progression in different ways. Adrenomedullin-1 receptor (AM1R) regulates blood pressure and blocking AM signaling via AM1R would be clinically unacceptable. Therefore, antagonizing adrenomedullin-2 receptor (AM2R) presents as an avenue for anti-cancer drug development. AREAS COVERED: We review the literature to highlight AM's role in cancer as well as delineating the specific roles AM1R and AM2R mediate in the development of a pro-tumoral microenvironment. We highlight the importance of exploring the residue differences between the receptors that led to the development of first-in-class selective AM2R small molecule antagonists. We also summarize the current approaches targeting AM and its receptors, their anti-tumor effects and their limitations. EXPERT OPINION: As tool compounds, AM2R antagonists will allow the dissection of the functions of CGRPR (calcitonin gene-related peptide receptor), AM1R and AM2R, and has considerable potential as a first-in-class oncology therapy. Furthermore, the lack of detectable side effects and good drug-like pharmacokinetic properties of these AM2R antagonists support the promise of this class of compounds as potential anti-cancer therapeutics.

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. EXPERIMENTAL APPROACH: Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. KEY RESULTS: An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. CONCLUSIONS AND IMPLICATIONS: RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors.

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.