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.

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure-Activity Relationships and Optimization.

Class B G-protein-coupled receptors (GPCRs) remain an underexploited target for drug development. The calcitonin receptor (CTR) family is particularly challenging, as its receptors are heteromers comprising two distinct components: the calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of three accessory proteins known as receptor activity-modifying proteins (RAMPs). CLR/RAMP1 forms a CGRP receptor, CLR/RAMP2 forms an adrenomedullin-1 (AM1) receptor, and CLR/RAMP3 forms an adrenomedullin-2 (AM2) receptor. The CTR/RAMP complexes form three distinct amylin receptors. While the selective blockade of AM2 receptors would be therapeutically valuable, inhibition of AM1 receptors would cause clinically unacceptable increased blood pressure. We report here a systematic study of structure-activity relationships that has led to the development of first-in-class AM2 receptor antagonists. These compounds exhibit therapeutically valuable properties with 1000-fold selectivity over the AM1 receptor. These results highlight the therapeutic potential of AM2 antagonists.

Accelerated Development With Increased Bone Mass and Skeletal Response to Loading Suggest Receptor Activity Modifying Protein-3 as a Bone Anabolic Target.

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression ß-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically.

Cross-Species RNA-Seq Study Comparing Transcriptomes of Enriched Osteocyte Populations in the Tibia and Skull.

Local site-specific differences between bones in different regions of the skeleton account for their different properties and functions. To identify mechanisms behind these differences, we have performed a cross-species study comparing RNA transcriptomes of cranial and tibial osteocytes, from bones with very different primary functions and physiological responses, collected from the same individual mouse, rat, and rhesus macaque. Bioinformatic analysis was performed to identify 32 genes changed in the same direction between sites and shared across all three species. Several well-established key genes in bone growth and remodeling were upregulated in the tibias of all three species (BMP7, DKK1, FGF1, FRZB, SOST). Many of them associate or crosstalk with the Wnt signaling pathway. These results suggest Wnt signaling-related candidates for different control of regulatory mechanisms in bone homeostasis in the skull and tibia and indicate a different balance between genetically determined structure and feedback mechanisms to strains induced by mechanical loading at the different sites.

Discovery of a First-in-Class Potent Small Molecule Antagonist against the Adrenomedullin-2 Receptor.

The hormone adrenomedullin has both physiological and pathological roles in biology. As a potent vasodilator, adrenomedullin is critically important in the regulation of blood pressure, but it also has several roles in disease, of which its actions in cancer are becoming recognized to have clinical importance. Reduced circulating adrenomedullin causes increased blood pressure but also reduces tumor progression, so drugs blocking all effects of adrenomedullin would be unacceptable clinically. However, there are two distinct receptors for adrenomedullin, each comprising the same G protein-coupled receptor (GPCR), the calcitonin receptor-like receptor (CLR), together with a different accessory protein known as a receptor activity-modifying protein (RAMP). The CLR with RAMP2 forms an adrenomedullin-1 receptor, and the CLR with RAMP3 forms an adrenomedullin-2 receptor. Recent research suggests that a selective blockade of adrenomedullin-2 receptors would be therapeutically valuable. Here we describe the design, synthesis, and characterization of potent small-molecule adrenomedullin-2 receptor antagonists with 1000-fold selectivity over the adrenomedullin-1 receptor, although retaining activity against the CGRP receptor. These molecules have clear effects on markers of pancreatic cancer progression in vitro, drug-like pharmacokinetic properties, and inhibit xenograft tumor growth and extend life in a mouse model of pancreatic cancer. Taken together, our data support the promise of a new class of anticancer therapeutics as well as improved understanding of the pharmacology of the adrenomedullin receptors and other GPCR/RAMP heteromers.

Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.

BACKGROUND AND AIM: The Receptor Activity Modifying Proteins (RAMPs) are a group of accessory proteins, of which there are three in humans, that interact with a number of G-protein coupled receptors (GPCR) and play various roles in regulation of endocrine signaling. Studies in RAMP3 knockout (KO) mice reveal an age related phenotype with altered metabolic regulation and high bone mass. To translate these findings into a clinically relevant perspective, we investigated the association between RAMP3 gene variants, body composition and bone phenotypes in two population-based cohorts of Swedish women. METHODS: Five single nucleotide polymorphisms (SNP) in the vicinity of the RAMP3 gene were genotyped in the PEAK-25 cohort (n = 1061; 25 years) and OPRA (n = 1044; 75 years). Bone mineral density (BMD), fat mass and lean mass (total body; regional) were measured by DXA at baseline, 5 and 10 year follow-up. RESULTS: BMD did not differ with RAMP3 genotype in either cohort, although fracture risk was increased in the elderly women (OR 2.695 [95% CI 1.514-4.801]). Fat mass tended to be higher with RAMP3 SNPs; although only in elderly women. In the young women, changes in BMI and fat mass between ages 25-35 differed by genotype (p = 0.001; p < 0.001). CONCLUSION: Variation in RAMP3 may contribute to age-related changes in body composition and risk of fracture.

Analysis of RAMP3 gene polymorphism with body composition and bone density in young and elderly women.

BACKGROUND AND AIM: The Receptor Activity Modifying Proteins (RAMPs) are a group of accessory proteins, of which there are three in humans, that interact with a number of G-protein coupled receptors (GPCR) and play various roles in regulation of endocrine signaling. Studies in RAMP3 knockout (KO) mice reveal an age related phenotype with altered metabolic regulation and high bone mass. To translate these findings into a clinically relevant perspective, we investigated the association between RAMP3 gene variants, body composition and bone phenotypes in two population-based cohorts of Swedish women. METHODS: Five single nucleotide polymorphisms (SNP) in the vicinity of the RAMP3 gene were genotyped in the PEAK-25 cohort (n = 1061; 25 years) and OPRA (n = 1044; 75 years). Bone mineral density (BMD), fat mass and lean mass (total body; regional) were measured by DXA at baseline, 5 and 10 year follow-up. RESULTS: BMD did not differ with RAMP3 genotype in either cohort, although fracture risk was increased in the elderly women (OR 2.695 [95% CI 1.514-4.801]). Fat mass tended to be higher with RAMP3 SNPs; although only in elderly women. In the young women, changes in BMI and fat mass between ages 25-35 differed by genotype (p = 0.001; p < 0.001). CONCLUSION: Variation in RAMP3 may contribute to age-related changes in body composition and risk of fracture.

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2).

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Role of receptor activity modifying protein 1 in function of the calcium sensing receptor in the human TT thyroid carcinoma cell line.

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca(2+) and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

Research resource: Haploinsufficiency of receptor activity-modifying protein-2 (RAMP2) causes reduced fertility, hyperprolactinemia, skeletal abnormalities, and endocrine dysfunction in mice.

Receptor activity-modifying protein-2 (RAMP2) is a single-pass transmembrane protein that can regulate the trafficking, ligand binding, and signaling of several G protein-coupled receptors (GPCR). The most well-characterized role of RAMP2 is in the regulation of adrenomedullin (AM) binding to calcitonin receptor-like receptor (CLR), and our previous studies using knockout mouse models support this canonical signaling paradigm. For example, Ramp2(-/-) mice die at midgestation with a precise phenocopy of the AM(-/-) and Calcrl(-/-) mice. In contrast, Ramp2(+/-) mice are viable and exhibit an expanded variety of phenotypes that are distinct from those of Calcrl(+/-) mice. Using Ramp2(+/-) female mice, we demonstrate that a modest decrease in Ramp2 expression causes severe reproductive defects characterized by fetal growth restriction, fetal demise, and postnatal lethality that is independent of the genotype and gender of the offspring. Ramp2(+/-) female mice also exhibit hyperprolactinemia during pregnancy and in basal conditions. Consistent with hyperprolactinemia, Ramp2(+/-) female mice have enlarged pituitary glands, accelerated mammary gland development, and skeletal abnormalities including delayed bone development and decreased bone mineral density. Because RAMP2 has been shown to associate with numerous GPCR, it is likely that signaling of one or more of these GPCR is compromised in Ramp2(+/-) mice, yet the precise identification of these receptors remains to be elucidated. Taken together, this work reveals an essential role for RAMP2 in endocrine physiology and provides the first in vivo evidence for a physiological role of RAMP2 beyond that of AM/CLR signaling.

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity.

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1(tr/tr)) have approximately 50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1(tr/tr) osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1(tr/tr) cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.