Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice.

BACKGROUND AND AIMS: Kinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs). METHODS: Fibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study. RESULTS: B1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFß, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-ß and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway. CONCLUSIONS: B1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

Patient Selection and Toxicities of PRRT for Metastatic Neuroendocrine Tumors and Research Opportunities.

OPINION STATEMENT: Neuroendocrine tumors (NETs) are a heterogenous group of neoplasms characterized by varied biological hallmarks and behavior, ranging from indolent to aggressive. For many decades, somatostatin analogues and few targeted therapies were available for NETs and these therapies had minimal response rates. However, there have been a number of recent treatment advances. Peptide receptor radionuclide therapy (PRRT) is a novel approach to treatment of NETs and has changed the landscape of treatment for NETs. It is a form of targeted therapy in which a radiolabeled somatostatin analogue delivers radiation specifically to tumor cells expressing the somatostatin receptor.

An Integrated Pan-Cancer Analysis and Structure-Based Virtual Screening of GPR15.

G protein-coupled receptor 15 (GPR15, also known as BOB) is an extensively studied orphan G protein-coupled receptors (GPCRs) involving human immunodeficiency virus (HIV) infection, colonic inflammation, and smoking-related diseases. Recently, GPR15 was deorphanized and its corresponding natural ligand demonstrated an ability to inhibit cancer cell growth. However, no study reported the potential role of GPR15 in a pan-cancer manner. Using large-scale publicly available data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we found that GPR15 expression is significantly lower in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) than in normal tissues. Among 33 cancer types, GPR15 expression was significantly positively correlated with the prognoses of COAD, neck squamous carcinoma (HNSC), and lung adenocarcinoma (LUAD) and significantly negatively correlated with stomach adenocarcinoma (STAD). This study also revealed that commonly upregulated gene sets in the high GPR15 expression group (stratified via median) of COAD, HNSC, LUAD, and STAD are enriched in immune systems, indicating that GPR15 might be considered as a potential target for cancer immunotherapy. Furthermore, we modelled the 3D structure of GPR15 and conducted structure-based virtual screening. The top eight hit compounds were screened and then subjected to molecular dynamics (MD) simulation for stability analysis. Our study provides novel insights into the role of GPR15 in a pan-cancer manner and discovered a potential hit compound for GPR15 antagonists.

Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4.

Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.

The prokineticin receptor antagonist PC1 rescues memory impairment induced by ß amyloid administration through the modulation of prokineticin system.

Growing evidences demonstrate that chemokines and chemokine receptors are up-regulated in resident central nervous system cells during Alzheimer's disease contributing to neuroinflammation and neurodegeneration. Prokineticin 2 belongs to a new family of chemokines which recently emerged as a critical player in immune system and inflammatory diseases. Since pharmacological blockade in vitro of the prokineticin system is able to antagonize Amyloid ß-induced neurotoxicity, the aim of the present study was to investigate in vivo effects of prokineticin receptor antagonist PC1 on memory impairment in a rodent model of Alzheimer's disease. Rats were intracerebroventricular infused with Aß1-42 and behavioral responses as well as the expression profile in hippocampus of prokineticin 2 and its receptors were investigated. Results demonstrated that Aß1-42-infused rats developed significant memory impairments together with a marked up-regulation of both prokineticin 2 and its receptors in hippocampal neurons and astrocytes. Treatment with PC1 significantly improved learning capability of Aß1-42-infused rats restoring the balance of prokineticin system. This study pointed to a neuroprotective role of PC1 on Aß1-42-induced memory deficits that could be ascribed to the ability of PC1 to modulate rat hippocampal prokineticin system and to recover the impaired Aß1-42-induced neurogenesis. This suggests that prokineticin system antagonism could be considered as a new approach for the study of AD etiopathology.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Biomedical applications of radioiodinated peptides.

The overexpression of peptide receptors in certain tumors as compared to endogeneous expression levels represents the molecular basis for the design of peptide-based tools for targeted nuclear imaging and therapy. Receptor targeting with radiolabelled peptides became a very important imaging and/or therapeutic approach in nuclear medicine and oncology. A great variety of peptides has been radiolabelled with clinical relevant radionuclides, such as radiometals and radiohalogens. However, to the best of our knowledge concise and updated reviews providing information about the biomedical application of radioiodinated peptides are still missing. This review outlines the synthetic efforts in the preparation of radioiodinated peptides highlighting the importance of radioiodine in nuclear medicine, giving an overview of the most relevant radioiodination strategies that have been employed and describes relevant examples of their use in the biomedical field.

Targeting Anthrax Toxin Receptor 2 Ameliorates Endometriosis Progression.

Rationale: Endometriosis is a highly prevalent gynecological disease in women of reproductive age that markedly reduces life quality and fertility. Unfortunately, there is no cure for this disease, which highlights that more efforts are needed to investigate the underlying mechanism for designing novel therapeutic regimens. This study aims to investigate druggable membrane receptors distinctively expressed in endometriotic cells. Methods: Bioinformatic analysis of public databases was employed to identify potential druggable candidates. Normal endometrial tissues and ectopic endometriotic lesions were obtained for the determination of target genes. Primary endometrial and endometriotic stromal cells as well as two different mouse models of endometriosis were used to characterize molecular mechanisms and therapeutic outcomes of endometriosis, respectively. Results: Anthrax toxin receptor 2 (ANTXR2) mRNA and protein are upregulated in the endometriotic specimens. Elevation of ANTXR2 promotes endometriotic cell adhesion, proliferation, and angiogenesis. Furthermore, hypoxia is the driving force for ANTXR2 upregulation via altering histone modification of ANTXR2 promoter by reducing the repressive mark, histone H3 lysine 27 (H3K27) trimethylation, and increasing the active mark, H3K4 trimethylation. Activation of ANTXR2 signaling leads to increased Yes-associated protein 1 (YAP1) nuclear translocation and transcriptional activity, which contributes to numerous pathological processes of endometriosis. Pharmacological blocking of ANTXR2 signaling not only prevents endometriotic lesion development but also causes the regression of established lesion. Conclusion: Taken together, we have identified a novel target that contributes to the disease pathogenesis of endometriosis and provided a potential therapeutic regimen to treat it.

The potent small molecule integrin antagonist THR-687 is a promising next-generation therapy for retinal vascular disorders.

Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4 ±â€¯2.7 nM), αvß5 (IC50 of 1.3 ±â€¯0.5 nM) and α5ß1 (IC50 of 6.8 ±â€¯3.2 nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258 ±â€¯113 nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236 ±â€¯173 nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45 mg, 2.25 mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.

Identification of the salusin-ß receptor using proteoliposomes embedded with endogenous membrane proteins.

Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-ß, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the ß-chain of ATP synthase is a cell surface receptor for salusin-ß by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a ß-actin-profilin complex as membrane-associated salusin-ß-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-ß modulates angiogenesis. Salusin-ß inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-ß binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.

Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.

In silico estrogen-like activity and in vivo osteoclastogenesis inhibitory effect of Cicer arietinum extract.

Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

RGD cadherins and α2ß1 integrin in cancer metastasis: A dangerous liaison.

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2ß1 integrin, these cadherins promote integrin ß1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2ß1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2ß1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2ß1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.

Targeting the Prokineticin System to Control Chronic Pain and Inflammation.

Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin- receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induces activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed.

Radiolabeled peptides: current and new perspectives.

Radiolabeled peptides have been the subject of research for over 20 years and during that time possibility/variety of peptide receptor imaging and later targeted radiotherapy increased significantly. The targeted receptors belong to the large family of G-protein-coupled receptors or tyrosine kinases receptors partially connected with them. They both regulate large signaling networks, control multiple cell functions and are implicated in many diseases including cancers. The essential feature of peptides used in nuclear medicine involves their ability to binding with high affinity and specify to their receptors overexpressed on tumor cells. Currently most important peptide radiotracers are somatostatin, GLP-1, bombesin, gastrin/cholecystokinin-2, neurokinin type 1, CXCR4, EGF, VEGF and integrins. These tracers are mainly based on nuclides which are radiometals or on 18F and may be used in both SPECT or PET techniques as well as for hybrid imaging. Using of chelators suitable for peptide labeling with diagnostic and therapeutic radionuclids enables the theranostic approach. In this review we will provide a brief overview over currently available radiopharmaceuticals based on different groups of peptides.

Role of relaxin-3/RXFP3 system in stress-induced binge-like eating in female rats.

Binge eating is frequently stimulated by stress. The neuropeptide relaxin-3 (RLN3) and its native receptor RXFP3 are implicated in stress and appetitive behaviors. We investigated the dynamics of the central RLN3/RXFP3 system in a newly established model of stress-induced binge eating. Female Sprague-Dawley rats were subjected to unpredictable intermittent 1-h access to 10% sucrose. When sucrose intake stabilized, rats were assessed for consistency of higher or lower sucrose intake in response to three unpredictable episodes of foot-shock stress; and assigned as binge-like eating prone (BEP) or binge-like eating resistant (BER). BEP rats displayed elevated consumption of sucrose under non-stressful conditions (30% > BER) and an additional marked increase in sucrose intake (60% > BER) in response to stress. Conversely, sucrose intake in BER rats was unaltered by stress. Chow intake was similar in both phenotypes on 'non-stress' days, but was significantly reduced by stress in BER, but not BEP, rats. After stress, BEP, but not BER, rats displayed a significant increase in RLN3 mRNA levels in the nucleus incertus. In addition, in response to stress, BEP, but not BER, rats had increased RXFP3 mRNA levels in the paraventricular and supraoptic nuclei of the hypothalamus. Intracerebroventricular administration of a selective RXFP3 antagonist, R3(B1-22)R, blocked the stress-induced increase in sucrose intake in BEP rats and had no effect on sucrose intake in BER rats. These results provide important evidence for a role of the central RLN3/RXFP3 system in the regulation of stress-induced binge eating in rats, and have therapeutic implications for eating disorders.

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.