Differential expression of components of the CGRP-receptor family in human coronary and human middle meningeal arteries: functional implications.

BACKGROUND: Different responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA. METHODS: RNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM22-52 and/or olcegepant. RESULTS: Calcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA. CONCLUSION: Based on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research.

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs.

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression.

Although the role of peripheral nerves in cancer progression has been appreciated, little is known regarding cancer/sensory nerve crosstalk and its contribution to bone metastasis and associated pain. In this study, we revealed that the cancer/sensory nerve crosstalk plays a crucial role in bone metastatic progression. We found that (i) periosteal sensory nerves expressing calcitonin gene-related peptide (CGRP) are enriched in mice with bone metastasis; (ii) cancer patients with bone metastasis have elevated CGRP serum levels; (iii) bone metastatic patient tumor samples express elevated calcitonin receptor-like receptor (CRLR, a CGRP receptor component); (iv) higher CRLR levels in cancer patients are negatively correlated with recurrence-free survival; (v) CGRP induces cancer cell proliferation through the CRLR/p38/HSP27 pathway; and (vi) blocking sensory neuron-derived CGRP reduces cancer cell proliferation in vitro and bone metastatic progression in vivo. This suggests that CGRP-expressing sensory nerves are involved in bone metastatic progression and that the CGRP/CRLR axis may serve as a potential therapeutic target for bone metastasis.

Quantitative proteomics reveals CLR interactome in primary human cells.

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine, and cancer. To date, the network of proteins interacting with CLR ("CLR interactome") in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation utilizing anti-human CLR antibody with label-free quantitative nano LC-MS/MS and quantitative in situ proximity ligation assay. By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ proximity ligation assay). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

Expression of Calcitonin Gene-Related Peptide and Calcitonin Receptor-like Receptor in Colorectal Adenocarcinoma.

Colorectal cancer is one of the most widespread types of cancer that still causes many deaths worldwide. The development of new diagnostic and prognostic markers, as well as new therapeutic methods, is necessary. The calcitonin gene-related peptide (CGRP) neuropeptide alongside its receptor calcitonin receptor-like receptor (CRLR) could represent future biomarkers and a potential therapeutic target. Increased levels of CGRP have been demonstrated in thyroid, prostate, lung, and breast cancers and may also have a role in colorectal cancer. At the tumor level, it acts through different mechanisms, such as the angiogenesis, migration, and proliferation of tumor cells. The aim of this study was to measure the level of CGRP in colorectal cancer patients' serum by enzyme-linked immunosorbent assay (ELISA) and determine the level of CGRP and CRLR at the tumor level after histopathological (HP) and immunohistochemical (IHC) analysis, and then to correlate them with the TNM stage and with different tumoral characteristics. A total of 54 patients with newly diagnosed colorectal adenocarcinoma were evaluated. We showed that serum levels of CGRP, as well as CGRP and CRLR tumor level expression, correlate with the TNM stage, with local tumor extension, the presence of lymph node metastasis, and distant metastasis, and also with the tumor differentiation degree. CGRP is present in colorectal cancer from the incipient TNM stage, with levels increasing with the stage, and can be used as a diagnostic and prognostic marker and may also represent a potentially new therapeutic target.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.

Acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (FLT3-ITD) and DNA methyltransferase 3A (DNMT3A) R882 double mutations had a worse prognosis compared with AML with FLT3-ITD or DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL) in AML with FLT3-ITD and DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITD and DNMT3A-R882 double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP). Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-C cells. An in vivo AML  animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosis and repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITD and DNMT3A-R882 double-mutated AML.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Expression profile of adrenomedullin and its specific receptors in liver tissues from patients with hepatocellular carcinoma and in tumorigenic cell line-secreted extracellular vesicles.

The transcriptional profile of adrenomedullin (AM), a new metastasis-related factor involved in hepatocellular carcinoma (HCC), and its specific receptors (CLR, RAMP1, RAMP3) were evaluated in liver tissues of HCV-positive HCC subjects undergoing liver transplantation (LR) and in donors (LD). AM and its specific receptor expression were also assessed in extracellular vesicles (EVs) secreted by tumorigenic (HepG2) and non-tumorigenic (WRL68) cells by Real-Time PCR. AM expression resulted significantly elevated in LR concerning LD (p = 0.0038) and, for the first time, significantly higher levels in HCC patients as a function of clinical severity (MELD score), were observed. RAMP3 and CLR expression increased in LR as a function of clinical severity while RAMP1 decreased. Positive correlations were found among AM, its receptors, and apoptotic markers. No AM mRNA expression difference was observed between HepG2 and WRL68 EVs. RAMP1 and RAMP3 resulted lower in HepG2 concerning WRL68 while significantly higher levels were observed for CLR. While results at tissue level characterize AM as a regulator of carcinogenesis-tumor progression, those obtained in EVs do not indicate AM as a target candidate, neither as a pathological biomarker nor as a marker involved in cancer therapy.

Expression of the Calcitonin Receptor-like Receptor (CALCRL) in Normal and Neoplastic Tissues.

Little information is available concerning protein expression of the calcitonin receptor-like receptor (CALCRL) at the protein level. Here, we developed a rabbit monoclonal antibody, 8H9L8, which is directed against human CALCRL but cross-reacts with the rat and mouse forms of the receptor. We confirmed antibody specificity via Western blot analyses and immunocytochemistry using the CALCRL-expressing neuroendocrine tumour cell line BON-1 and a CALCRL-specific small interfering RNA (siRNA). We then used the antibody for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic tissues. In nearly all tissue specimens examined, CALCRL expression was detected in the capillary endothelium, smooth muscles of the arterioles and arteries, and immune cells. Analyses of normal human, rat, and mouse tissues revealed that CALCRL was primarily present in distinct cell populations in the cerebral cortex; pituitary; dorsal root ganglia; epithelia, muscles, and glands of the larger bronchi; intestinal mucosa (particularly in enteroendocrine cells); intestinal ganglia; exocrine and endocrine pancreas; arteries, capillaries, and glomerular capillary loops in the kidneys; the adrenals; Leydig cells in the testicles; and syncytiotrophoblasts in the placenta. In the neoplastic tissues, CALCRL was predominantly expressed in thyroid carcinomas, parathyroid adenomas, small-cell lung cancers, large-cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear-cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours with strong expression of CALCRL, the receptor may represent a useful target structure for future therapies.

The CGRP Receptor Antagonist MK0974 Induces EVI1high AML Cell Apoptosis by Disrupting ERK Signaling.

BACKGROUND/AIM: Acute myeloid leukemia (AML) with high expression of the oncogenic transcription factor ecotropic viral integration site-1 (EVI1) (EVI1high AML) is refractory, and there is an urgent need to develop treatment for EVI1high AML. We previously showed that calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein 1 (RAMP1) is highly expressed in EVI1high AML and participates in calcitonin gene-related peptide (CGRP)-induced stress hematopoiesis. This study examined whether MK0974 (a CGRP antagonist) acts as a therapeutic agent in CRLR/RAMP1high AML cell lines. MATERIALS AND METHODS: An in vitro experimental system was used to determine the effect of MK0974 on EVI1high AML cell lines. The expression of CRLR and RAMP1-3 in EVI1high and EVI1low AML lines was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). Next, MK0974 was added to the AML cell lines, and cell proliferation, cell cycle and apoptosis assays were carried out using flow cytometry (FCM). Proteins were evaluated using western blot analysis. We also generated AML cell lines with CRLR knockdown and evaluated whether the effect of MK0974 was reduced. RESULTS: Apoptosis was induced by adding MK0974 to the EVI1high AML cell line. In the EVI1high AML cell line, the addition of MK0974 attenuated the phosphorylation of ERK and p38. These effects were also attenuated by CRLR knockdown. CONCLUSION: MK0974, a CGRP receptor antagonist, inhibits the CRLR/RAMP1 complex and induces apoptosis, making it a potential therapeutic agent for CRLR/RAMP1high AML.

Plasma Clearance of Intravenously Infused Adrenomedullin in Rats with Acute Renal Failure.

Plasma adrenomedullin concentrations are reportedly elevated in patients with renal failure; however, the underlying mechanism is unclear. In this study, we investigated the plasma clearance of synthetic human adrenomedullin (AM) in two models of rats with renal dysfunction; one was induced by subcutaneous injection of mercury chloride (RD-Ag) and the other by completely blocking bilateral renal blood flow (RD-Bl). Sixty minutes after starting intravenous AM infusion, AM levels in RD-Ag, RD-Bl, and rats with normal renal function (NF) were still increased slightly; however, plasma AM levels in RD-Ag rats were approximately three times as high as in RD-Bl and NF rats. Plasma AM disappearance after the end of treatment was similar among the three groups. Pharmacokinetic analysis revealed that elevated plasma AM in RD-Ag rats may be caused by a reduced volume of distribution. The adrenomedullin functional receptor is composed of heterodimers, including GPCR, CLR (calcitonin receptor-like receptor, CALCRL), and the single transmembrane proteins, RAMP2 or RAMP3 (receptor activity modifying protein). Calcrl expression was downregulated in the lungs and kidneys of RD-Ag rats. Furthermore, the plasma concentration of exogenous AM was elevated in mice deficient in vascular endothelium-specific Ramp2. These results suggest that decreased plasma AM clearance in RD-Ag is not due to impaired renal excretion but to a decreased volume of distribution caused by a reduction in adrenomedullin receptors.

Procalcitonin mediates vascular dysfunction in obesity.

AIMS: Obesity is accompanied by a chronic low-grade inflammation associated with endothelial dysfunction and vascular complications. Procalcitonin is a marker of inflammation, secreted by adipose tissue and elevated in obese subjects. We here investigated whether visceral or perivascular fat-derived procalcitonin is a target to improve obesity-induced endothelial dysfunction. MATERIALS AND METHODS: Procalcitonin expression was identified by Western blot. Murine endothelial cells were isolated using CD31-antibody-coated magnetic beads and reactive oxygen species and nitric oxide (NO) determined by H2DCF- or DAF-FM diacetate loading. Endothelium-dependent vasorelaxation was analyzed using pressure myography of murine arterioles. Calcitonin gene-related peptide (CGRP) was used to activate the calcitonin receptor-like receptor (CRLR)/RAMP1 complex and olcegepant or the dipeptidyl-peptidase 4 (DPP4) inhibitor sitagliptin to block procalcitonin signaling or activation. KEY FINDINGS: In addition to visceral adipose tissue, procalcitonin was present in perivascular and epicardial tissue. In concentrations typical for obesity, procalcitonin doubled reactive oxygen species formation and decreased endothelial nitric oxide production in murine endothelial cells. Intravenous delivery of procalcitonin to mice in obesity-associated concentrations impaired endothelium-dependent vasorelaxation in a CRLR/RAMP1-dependent manner and antagonized CGRP-induced endothelial NO release in vitro. Use of CRLR/RAMP1-receptor antagonist olcegepant counteracted procalcitonin effects on vasodilation, nitric oxide production and reactive oxygen species formation. Similarly, blocking procalcitonin activation by the DPP4 inhibitor sitagliptin antagonized endothelial procalcitonin effects. SIGNIFICANCE: Procalcitonin, liberated either from visceral or perivascular adipose tissue, contributes to endothelial dysfunction by antagonizing CGRP signaling in obesity. Targeting hyperprocalcitonemia may be a means to preserve endothelial function and reduce comorbidity burden in obese subjects.

Profiling prediction of nuclear receptor modulators with multi-task deep learning methods: toward the virtual screening.

Nuclear receptors (NRs) are ligand-activated transcription factors, which constitute one of the most important targets for drug discovery. Current computational strategies mainly focus on a single target, and the transfer of learned knowledge among NRs was not considered yet. Herein we proposed a novel computational framework named NR-Profiler for prediction of potential NR modulators with high affinity and specificity. First, we built a comprehensive NR data set including 42 684 interactions to connect 42 NRs and 31 033 compounds. Then, we used multi-task deep neural network and multi-task graph convolutional neural network architectures to construct multi-task multi-classification models. To improve the predictive capability and robustness, we built a consensus model with an area under the receiver operating characteristic curve (AUC) = 0.883. Compared with conventional machine learning and structure-based approaches, the consensus model showed better performance in external validation. Using this consensus model, we demonstrated the practical value of NR-Profiler in virtual screening for NRs. In addition, we designed a selectivity score to quantitatively measure the specificity of NR modulators. Finally, we developed a freely available standalone software for users to make profiling predictions for their compounds of interest. In summary, our NR-Profiler provides a useful tool for NR-profiling prediction and is expected to facilitate NR-based drug discovery.

Virtual drug repurposing study for the CGRPR identifies pentagastrin and leuprorelin as putative candidates.

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer consisting of CLR and RAMP1 proteins. Activation of the CGRPR with the endogenous peptide CGRP is known to play a crucial role in migraine pathophysiology. CGRP occupies two regions in the CGRPR upon binding, namely ectodomain and transmembrane sites (sites 1 and 2, respectively). The disruption of the CGRPR heterodimer interface is one of the main strategies to prevent CGRPR activation and its resulting effects. So far, FDA approved monoclonal antibodies and small molecule gepant inhibitors are considered for the treatment of acute or chronic migraine symptoms. However, most of these gepants have severe side effects. Thus, in this study, a virtual drug repurposing approach is applied to CGRPR to find alternative or better molecules that would have a potential to inhibit or block the CLR - RAMP1 interface compared to known gepant molecules. A small molecule library of FDA-approved molecules was screened in these two different binding sites, further simulations were performed and analyzed. The objectives of this study are (i) to repurpose an FDA-approved drug having more potent features for CGRPR inhibition compared to gepants, and (ii) to examine whether the transmembrane binding site (site 2) accepts small molecules or small peptide analogues for binding. As a result of this extensive in silico analysis, two molecules were identified, namely pentagastrin and leuprorelin. It is shown that FDA approved compound rimegepant and the identified pentagastrin molecules form and maintain the interactions through CLR W72 and RAMP1 W74, which are the residues revealed to have an important role in CGRPR antagonism at binding site 1. At binding site 2, the interactions needed to be formed for CGRP binding are not captured by rimegepant nor leuprorelin, yet leuprorelin forms more interactions throughout the simulations, meaning that small molecules are also capable of binding to site 2. Moreover, it is found that the crucial interactions for receptor signaling and heterodimerization occurred between CLR and RAMP1 interface are disrupted more with the ligands bound to ectodomain site, rather than the transmembrane domain. These findings of pentagastrin and leuprorelin molecules are recommended to be considered in further de novo drug development and/or experimental studies related to CGRPR signaling blockade and antagonism.

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.

Novel Macrocyclic Antagonists of the Calcitonin Gene-Related Peptide Receptor: Design, Realization, and Structural Characterization of Protein-Ligand Complexes.

A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 (1) and HTL0028125 (2), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure-activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.

Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice.

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Peptide ligand interaction with maltose-binding protein tagged to the calcitonin gene-related peptide receptor: The inhibitory role of receptor N-glycosylation.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor-like receptor (CLR) with the different types of receptor activity-modifying protein (RAMP). Several crystal structures of the CGRP and AM receptor extracellular domain (ECD) used maltose-binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N-terminal tag MBP located in proximity of bound/mutated peptide ligands. This study provided evidence that MBP N-terminally tagged to the CGRP receptor ECD formed chemical interaction with the mutated peptide ligands. Interestingly, N-glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. I found that the N-glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N-glycosylation of the CGRP receptor ECD inhibited the tag protein interaction suggesting an additional function of N-glycosylation in the MBP-fused CGRP receptor ECD. This study reveals the importance of using tag protein-free versions of the CGRP receptor for the accurate assessment of peptide binding affinity.