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Supplementary figures 1 through 8.

Supplementary Table 1

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 –KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain.

Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity and glucose tolerance, wherein smaller adipocytes are insulin-sensitive and larger adipocytes develop insulin resistance and exhibit an impaired glucose uptake. Thus, pharmacological strategies aimed at regulating adipocyte hypertrophy (increase in adipocyte size) in favor of promoting hyperplasia (increase in adipocyte number) have the potential to improve adipocyte insulin sensitivity and provide therapeutic benefits in the context of metabolic disorders. As white adipose tissue can metabolize large amounts of glucose to lactate, using transcriptomics and in vitro characterization we explore the functional consequences of inhibiting monocarboxylate transporter 1 (MCT1) activity in fully differentiated adipocytes. Our studies show that the pharmacological inhibition of MCT1, a key regulator of the cellular metabolism and proliferation, promotes the re-entry of mature adipocytes into the cell cycle. Furthermore, we demonstrate that inhibitor-treated adipocytes exhibit an enhanced insulin-stimulated glucose uptake as compared with untreated adipocytes, and that this outcome is dependent on the cyclin-dependent kinase 1 (CDK1) activity. In summary, we identify a mechanism though which MCT1 inhibition improves the insulin sensitivity of mature adipocytes by inducing cell cycle re-entry. These results provide the foundation for future studies investigating the role MCT1 plays in adipocyte hyperplasia, and its therapeutic potential as a drug target for obesity and metabolic disease.

Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC(10) of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.

Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

The orexin 1 receptor (OX1R) and the kappa opioid receptor (KOR) are two G protein-coupled receptors (GPCRs) previously demonstrated to play important roles in modulating the rewarding effects of drugs of abuse such as cocaine. Using cells heterologously expressing both receptors, we investigated whether OX1R can regulate the function of KOR and vice versa. Activation of OX1R was found to attenuate agonist-activated KOR-mediated inhibition of cAMP production. In contrast, agonist-activated KOR-mediated β-arrestin recruitment and p38 activation were enhanced in the presence of activated OX1R. These effects are independent of OX1R internalization but are blocked in the presence of the JNK inhibitor SP-600125. OX1R signaling does not affect ligand binding by KOR. Taken together, these data suggest that OX1R signaling can modulate KOR function in a JNK-dependent manner, promoting preferential signaling of KOR via β-arrestin/p38 rather than Gαi. Conversely, Gαq coupling of OX1R is unaffected by activation of KOR, suggesting that this crosstalk is unidirectional. Given that KOR Gαi-mediated signaling events and β-arrestin-mediated signaling events are thought to promote distinct cellular responses and physiological outcomes downstream of KOR activation, this mechanism may have important implications on the behavioral effects of KOR activity.

Positive modulation of the GABAB receptor (GABABR) represents a potentially useful therapeutic approach for the treatment of nicotine addiction. The positive allosteric modulators (PAMs) of GABABR GS39783 and BHF177 enhance GABA‐stimulated [35S]GTP γS‐binding, and have shown efficacy in a rodent nicotine self‐administration procedure reflecting aspects of nicotine dependence. Interestingly, the structural related analog, NVP998, had no effect on nicotine self‐administration in rats despite demonstrating similar pharmacokinetic properties. Extensive in vitro characterization of GS39783, BHF177, and NVP998 activity on GABABR‐regulated signaling events, including modulation of cAMP, intracellular calcium levels, and ERK activation, revealed that these structurally related molecules display distinct pathway‐specific signaling activities that correlate with the dissimilarities observed in rodent models and may be predictive of in vivo efficacy. Furthermore, these GABABR allosteric modulators exhibit species‐dependent activity. Collectively, these data will be useful in guiding the development of GABABR allosteric modulators that display optimal in vivo efficacy in a preclinical model of nicotine dependence, and will identify those that have the potential to lead to novel antismoking therapies.

The galanin 3 receptor (GalR3) belongs to the large G protein-coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening-compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. DOI: http://dx.doi.org/10.7554/eLife.13662.001, eLife digest Calcium ions regulate many processes in the human body. The calcium-sensing receptor, called CaSR, is responsible for maintaining a stable level of calcium ions in the blood. This receptor can detect small changes in the concentration of calcium ions, and activates signalling events within the cell to restore the level of calcium ions back to normal. Abnormal activity of this receptor is associated with severe diseases in humans CaSR is found in the surface membrane of cells and belongs to a family of proteins called G-protein coupled receptors. Much of the protein extends out of the cell and interacts with calcium ions, phosphate ions and certain other molecules such as amino acids. However, it was not well understood how these small molecules bind to CaSR and how this activates the receptor. Geng et al. have now used a technique called X-ray crystallography to view the three-dimensional structure of the exterior domain of CaSR in its resting state and active state. These structures revealed that, contrary to expectations, calcium ions are not the main activator of the receptor. Instead, Geng et al. found that CaSR adopts an inactive state in the absence or presence of calcium ions, while the active state only forms when an amino acid is bound. Furthermore investigation showed that calcium ions are needed to stabilise the active form, while phosphate ions keep the inactive form stable. Geng et al. also identified the shape changes that must occur as CaSR transitions from its inactive to its active state. In particular, an amino acid binding to the exterior domain causes it to close like a venus flytrap, which is a crucial step in activating the receptor. Taken together, the findings show that the amino acids and calcium ions act jointly to fully activate CaSR. The next steps are to determine the structure of the entire receptor with and without its small molecule partners and to use these structures to design drugs that can alter CaSR’s activity in order to treat human diseases. DOI: http://dx.doi.org/10.7554/eLife.13662.002

A series of orexin receptor antagonists was synthesized based on a substituted piperidine scaffold. Through traditional medicinal chemistry structure–activity relationships (SAR), installation of various groups at the 3–6-positions of the piperidine led to modest enhancement in receptor selectivity. Compounds were profiled in vivo for plasma and brain levels in order to identify candidates suitable for efficacy in a model of drug addiction.

The central nervous melanocortin system forms a neural network that maintains energy homeostasis. Actions involving neural melanocortin-3 receptors (MC3Rs) regulate the expression rhythms in ingestive behaviors and metabolism anticipating nutrient intake. Here, we characterized the response of Mc3r knockout (Mc3r(-/-)) and wild type (WT) mice to a restricted feeding (RF) schedule where food access was limited to a 4-h period mid light cycle using a mechanical barrier. Mc3r(-/-) mice adapted poorly to the food restriction schedule. Anticipatory activity and the initial bout of intense feeding activity associated with granting food access were attenuated in Mc3r(-/-) mice, resulting in increased weight loss relative to controls. To investigate whether activity in specific hypothalamic nuclei contribute to the Mc3r(-/-) phenotype observed, we assessed hypothalamic FOS-immunoreactivity (FOS-IR) associated with food restriction. Food access markedly increased FOS-IR in the dorsomedial hypothalamus (DMH), but not in the suprachiasmatic or ventromedial hypothalamic nuclei (SCN and VMN, respectively) compared to ad libitum fed mice. Mc3r(-/-) mice displayed a significant reduction in FOS-IR in the DMH during feeding. Analysis of MC3R signaling in vitro indicated dose-dependent stimulation of the extracellular signal-regulated kinase (ERK) pathway by the MC3R agonist d-Trp(8)-γMSH. Treatment of WT mice with d-Trp(8)-γMSH administered intracerebroventricularly increased the number of pERK neurons 1.7-fold in the DMH. These observations provide further support for the involvement of the MC3Rs in regulating adaptation to food restriction. Moreover, MC3Rs may modulate the activity of neurons in the DMH, a region previously linked to the expression of the anticipatory response to RF.

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion induction, a steroid hormone that contributes to the pathology of postmyocardial infarction (MI) heart failure (HF), is mediated by both Gq/11 proteins and β‐arrestins, both of which couple to the AngII type 1 receptors (AT1Rs) of adrenocortical zona glomerulosa (AZG) cells. Over the past several years, AngII analogs with increased selectivity (“bias”) toward β‐arrestin‐dependent signaling at the AT1R have been designed and described, starting with SII, the gold‐standard β‐arrestin‐”biased” AngII analog. In this study, we examined the relative potencies of an extensive series of AngII peptide analogs at relative activation of G proteins versus β‐arrestins by the AT1R. The major structural difference of these peptides from SII was their varied substitutions at position 5, rather than position 4 of native AngII. Three of them were found biased for β‐arrestin activation and extremely potent at stimulating aldosterone secretion in AZG cells in vitro, much more potent than SII in that regard. Finally, the most potent of these three ([Sar1, Cys(Et)5, Leu8]‐AngII, CORET) was further examined in post‐MI rats progressing to HF and overexpressing adrenal β‐arrestin1 in vivo. Consistent with the in vitro studies, CORET was found to exacerbate the post‐MI hyperaldosteronism, and, consequently, cardiac function of the post‐MI animals in vivo. Finally, our data suggest that increasing the size of position 5 of the AngII peptide sequence results in directly proportional increases in AT1R‐dependent β‐arrestin activation. These findings provide important insights for AT1R pharmacology and future AngII‐targeted drug development.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). GLP-1R signals through G-protein-dependent, and G-protein-independent pathways by engaging the scaffold protein β-arrestin; preferential signalling of ligands through one or the other of these branches is known as ‘ligand bias'. Here we report the discovery of the potent and selective GLP-1R G-protein-biased agonist, P5. We identified P5 in a high-throughput autocrine-based screening of large combinatorial peptide libraries, and show that P5 promotes G-protein signalling comparable to GLP-1 and Exendin-4, but exhibited a significantly reduced β-arrestin response. Preclinical studies using different mouse models of T2DM demonstrate that P5 is a weak insulin secretagogue. Nevertheless, chronic treatment of diabetic mice with P5 increased adipogenesis, reduced adipose tissue inflammation as well as hepatic steatosis and was more effective at correcting hyperglycaemia and lowering haemoglobin A1c levels than Exendin-4, suggesting that GLP-1R G-protein-biased agonists may provide a novel therapeutic approach to T2DM., GLP-1 is a gut hormone with glucose-lowering activity. Here the authors report the peptide, P5, a variant of the GLP-1 receptor agonist exendin-4, with 'biased' signalling activity, and show that P5 improves glucose homeostasis in diabetic mice by increasing adipose tissue hyperplasia.

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion is mediated by either Gq/11 proteins or βarrestin1 (βarr1), both of which can couple to its type 1 receptors (AT1Rs), present in adrenocortical zona glomerulosa (AZG) cell membranes. In the present study, we examined the relative potencies of all the currently used in the clinic AT1R antagonist drugs (angiotensin receptor blockers, ARBs, or sartans) at preventing activation of these two signaling mediators (G proteins and βarrs) at the AngII-bound AT1R and, consequently, at suppression of aldosterone in vitro. All ARBs were found to be potent inhibitors of G protein activation at the AT1R. However, candesartan and valsartan were the most potent at blocking AngII-induced βarr activation at this receptor, among the tetrazolo-biphenyl-methyl derivatives, translating into excellent efficacies at aldosterone suppression in H295R cells. Conversely, irbesartan and losartan were largely G protein-selective inhibitors at the AT1R, with very low potency towards βarr inhibition. As a result, they were very weak suppressors of βarr1-dependent aldosterone production in H295R cells. These findings provide important pharmacological insights into the drug class of ARBs and medicinal chemistry insights for future drug development in the field of AngII antagonism.

AimsThis study evaluated a framework for modelling the continuous exchange of carbon (C) between the atmosphere, plants, humus, and microorganisms, proposing a plant C model coupled to MOMOS, an existing microbial C model.MethodsC data were collected on low fertility cereal-legume cropping systems. Plant C and microbial C were modelled simultaneously and the growth parameters of plants and nitrogen-fixing microorganisms were fitted to the data.ResultsAll C exchanges were successfully predicted using the same weather correction for plant and microbial processes. Most of the photosynthetic production was allocated to the roots, reducing yields. The C losses were found modelled mainly by root respiration for cereals, probably as an energy source for nutrient explorings, and by root mortality for legumes as a growth source for decomposers and symbiotic nodules. The effect of root-nodule activity on shoot growth was found non-linear. The system was modelled as a sink of 4.2 Mg C ha-1 year−1 in the soil’s labile C reserve.ConclusionsThis paper coordinates theoretical bases for modelling the processes regulating plant productivity associated with plant C losses. The tool appears to be robust and is now available for calculating the essential parameters of agro-ecology and climate change.

Introduction: The scaffolding protein βarrestin1 (βarr1) by the angiotensin II (AngII) type 1 receptor (AT 1 R) mediates AngII-induced aldosterone production in vitro and physiologically in vivo, thereby exacerbating heart failure (HF) progression post-myocardial infarction (MI). Herein, we sought to investigate the relative potency of various AT 1 R antagonist drugs (sartans) at inhibiting βarr vs. G protein activation and hence aldosterone production in vitro and in vivo. We also investigated the alterations in plasma aldosterone levels conferred by these agents and their impact on cardiac function of post-MI rats. Methods: For the in vitro tests, transfected CHO and adrenocortical H295R cells were used. For in vivo studies, post-MI rats overexpressing βarr1 in their adrenals received 7-day-long treatments with the drugs of interest. Results: Among the sartans tested, candesartan and valsartan were the most potent βarr activation and βarr-mediated aldosterone production inhibitors in vitro, as well as the most “biased” antagonists towards βarr vs. G-protein inhibition. Conversely, losartan and irbesartan were the least potent βarr inhibitors and the least “biased” antagonists towards βarr inhibition. These in vitro findings were corroborated in vivo, since candesartan and valsartan, contrary to irbesartan, caused significant plasma aldosterone reductions in post-MI rats. Accordingly, cardiac ejection fraction (EF) and contractility were significantly augmented in candesartan- and valsartan-treated rats (EF: 41.1±1% and 40±1% respectively, vs. 35±0.3% for saline-treated), but further deteriorated in irbesartan-treated post-MI rats (EF: 32±1%, n=7 rats/group). Conclusions: These findings provide important insights that might aid pharmacotherapeutic decisions (i.e. individual agent selections) involving this commonly prescribed cardiovascular drug class (sartans).

Differentiating somatic from emotional influences on the experience of chronic pain has been of interest to clinicians and researchers for many years. Although prior research has not well specified these pathways at the anatomical level, some evidence, both theoretical and empirical, suggest that emotional reactions influence the experience of disease and non-disease-related pains. Other studies suggest that treatments directed at negative emotional responses reduce suffering associated with pain. The current study was conducted to explore the influence of emotional reactions to pain as a predictor of psychological distress in a sample of adult Blacks with Sickle Cell Disease (SCD). Using cross-sectional survey data, we evaluated whether negative emotional reactions to the experience of pain were predictive of psychological distress after controlling for the somatic dimension of pain and age in n = 67 Black patients with Sickle Cell Disease (SCD). Results showed that greater negative emotion associated with pain predicted Somatization ( p < .01), Anxiety ( p < .05), Phobic Anxiety ( p < .05), and Psychoticism ( p < .05). Increased negative emotion associated with pain was also predictive of the General Symptoms Index ( p < .05) and the Positive Symptoms Total from the SCL-90-R ( p < .01). We believe the current study demonstrates that negative emotional reactions to the experience of pain in adults with SCD are predictive of psychological distress above and beyond the influences of age and the direct nociceptive experience. We also believe these data to be valuable in conceptualizing the allocation of treatment resources toward a proactive approach with early identification of patients who are responding poorly for the purpose of potentially reducing later psychopathology. A deeper understanding of the ways that subpopulations cope with chronic disease-related pain may produce models that can be ultimately generalized to the consumers of the majority of healthcare resources.

Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson’s disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca2+ mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2 μM at NTSR1 with full agonist behaviour (Emax = 100%), showing a higher efficacy and ~ 90-fold potency improvement compared to parent compound 1 (EC50 = 178 μM; Emax = 17%).

GABAB receptors (GABABR) are thought to be one of the most promising targets for the treatment of drug dependence. GABABR positive allosteric modulators (PAMs) have been shown to exhibit similar ef...

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of beta2-adrenergic receptors (beta2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human beta2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the beta2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the beta2-AR agonist, zinterol. Treatment with the beta2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean beta-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 +/- 286 versus beta2-AR, 6250.6* +/- 432.5 mmHg/s; n = 10, *P < 0.02). beta2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of beta2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

Attenuated activity of the central nervous melanocortin system causes obesity and insulin resistance. Obese rodents treated with melanocortins exhibit improvements in obesity and metabolic homeostasis that are not mutually dependent, suggesting metabolic actions that are independent of weight changes. These responses are generally thought to involve G-protein-coupled receptors expressed in the brain. Melanocortin-4 receptors (MC4Rs) regulate satiety and autonomic nervous system and thyroid function. MC3Rs are expressed in hypothalamic and limbic regions involved in controlling ingestive behaviors and autonomic function. Mc3r−/− mice exhibit increased adiposity and an accelerated diet-induced obesity. While this phenotype is not dependent on hyperphagia, data on the regulation of food intake by MC3Rs are inconsistent. Recent investigations by our laboratory suggest a unique combination of behavioral and metabolic disorders in Mc3r−/− mice. MC3Rs are critical for the expression of the anticipatory response and metabolic homeostasis when food intake occurs outside the normal voluntary rhythms driven by photoperiod. Using a Cre-Lox strategy, we can now investigate MC3Rs expressed in different brain regions and organ systems in the periphery. While focusing on the functions of neural MC3Rs, early results suggest an additional layer of complexity with central and peripheral MC3Rs involved in the defense of body weight.

We analyzed the medical records of all live birth infants (LBs) born with major birth defects (MBDs) at King Fahd Hofuf Hospital (KFHH) during a three-year period. Our objectives were to determine defect prevalence rate, patterns, geographic differences, associated maternal and infant risk factors, and the contribution of the defects to mortality in our Neonatal Intensive Care Unit (NICU). Out of 30,159 infants born alive during the study period, 687 (2.27%) had one or more MBDs. Systems most commonly affected were the cardiac (20.8%), musculoskeletal (18.7%), and central nervous (18.3%) systems. We observed higher rates/1000 LBs of life-threatening CNS and cardiac defects and diaphragmatic hernia than rates reported from other countries. Rate of defects/1000 LBs increased from 0.79% in the birth-weight group/=4000 g to 15.2% in the birth-weight category1500 g. Fifty-six infants with MBDs were born to diabetic mothers; diabetes was the only identified maternal disease associated with birth defects. Diabetic mothers and those of infants with chromosomal anomalies had higher means of age and parity than the control mothers ( 33.5 yrs (SD 5.2), 31.4 yrs (7.5), and 8.8 (3.8), 7.39 (3.8), for age and parity of diabetic and chromosomal anomalies respectively vs. 26.8 yrs (6.4) and 5.2 (3.7) in the controls, P0.01). Out of the 687 infants, 254 (36.97%) died; and MBDs were the most common disease- specific cause of death in our NICU throughout the study period.

The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress.

The apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed serine/threonine protein kinase and one of more than 20 members that make up the triple MAP kinase (MAP3K) family of enzymes. Over the past decade, genetic studies have revealed that ASK1 plays a pivitol role in the cellular response to a wide variety of environmental and biological stressors including; reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress caused by protein aggregation, influx of calcium ions, and receptor-mediated signals transduced via lipopolysaccharides (LPS), Fas ligand, cytokines (TNFα) and certain G protein-coupled receptor (GPCR) agonists [1-5]. In addition, exogenous expression of ASK1 in cells has shown that ASK1 signaling engages the intrinsic apoptosis pathway promoting cytochrome c release from mitochodria and subsequent activation of caspase 3 and 9 [1, 6, 7]. Conversely, ASK1 deficient cells are resistant to cell death induced by oxidative and ER stress, indicating that ASK1 acts as the lynch pin in certain forms of stress-induced cell death [8]. Once activated, ASK1 relays cellular stress signals via the classical three tierd mitogen activated protein kinase (MAPK) signaling cascade, whereby a MAP3K phosphorylates and activates a MAP2K, that in turn phosphorylates and activates a MAPK [9] (Figure 1). More specifically, the ASK1 signaling axis activates the p38 and the c-jun NH2-terminal kinases (JNK) family of MAPKs, via activation of MKK3/MKK6 and MKK4/MKK7 respectively [1, 2, 4]. In addition to its role in the cellular stress response, ASK1 also regulates physiological processes including neuronal differentiation, synaptic plasticity and the innate immune response [10-13]. Thus, ASK1 acts as an important regulator of several important biological processess and not surprisingly, ASK1 activation is under tight regulatory control. Regulation of ASK1 activity is accomplished via a number of mechanisms including; protein-protein interactions as well as both spatial and temporal control. Firstly, more than 30 ASK1 interacting partners have been shown to regulate ASK1 activity (either positively or negatively) by posttranslational modifications and/or by inducing conformational changes through protein-protein interactions. Secondly, ASK1 signaling complexes are located in both the cytoplasm and mitochondria [14], with nuclear translocation observed upon stress induction indicating that ASK1 localization might also dictate the biological outcome [15,16] and thirdly, duration of ASK1 signaling can influence the nature of the

G protein-coupled receptors (GPCR) are the major site of action for endogenous hormones and neurotransmitters. Early drug discovery efforts focused on determining whether ligands could engage G protein coupling and subsequently activate or inhibit cognate "second messengers." Gone are those simple days as we now realize that receptors can also couple βarrestins. As we delve into the complexity of ligand-directed signaling and receptosome scaffolds, we are faced with what may seem like endless possibilities triggered by receptor-ligand mediated events.

The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC50 values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (Ki values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.

Heme-oxygenase-1 (HO-1), a stress-inducible protein, is an important cytoprotective agent against ischemia/reperfusion (I/R) injury. However, the role of downstream mediators involved in HO-1-induced cytoprotection is not clear. In the current study we investigated the role of biliverdin reductase, an enzyme involved in the conversion of HO-1-derived biliverdin into bilirubin and the PI3K/Akt pathway in mediating the cytoprotective effects of HO-1 against hypoxia and reoxygenation (H/R) injury in vitro and in vivo. H9c2 cardiomyocytes were transfected with a plasmid expressing HO-1 or LacZ and exposed to 24 h of hypoxia followed by 12 h of reoxygenation. At the end of reoxygenation, reactive oxygen species generation was determined using CM-H(2)DCFDA dye and apoptosis was assessed by TUNEL, caspase activity and Bad phosphorylation. p85 and Akt phosphorylation were determined using cell-based ELISA and phospho-specific antibodies, respectively. HO-1 overexpression increased phosphorylation of the regulatory subunit of the PI3K (p85alpha) and downstream effector Akt in H9c2 cells, leading to decreased ROS and apoptosis. Furthermore, cardiac expression of HO-1 increased basal phosphorylated Akt levels and decreased infarct size in response to LAD ligation and release induced I/R injury. Conversely, PI3K inhibition reversed the effects of HO-1 on Akt phosphorylation, cell death and infarct size. In addition, knockdown of biliverdin reductase (BVR) expression with siRNA attenuated HO-1-induced Akt phosphorylation and increased H/R-induced apoptosis of H9c2 cells. Co-immunoprecipitation revealed protein-protein interaction between BVR and the phosphorylated p85 subunit of the PI3 kinase. Taken together, these results suggest that the enzyme biliverdin reductase plays an important role in mediating cytoprotective effects of HO-1. This effect is mediated, at least in part, via interaction with and activation of the PI3K/Akt pathway.

Background Stimulation of β 1 - and β 2 -adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the β 3 AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. Methods and Results To better understand the role of β 3 ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human β 3 AR (TGβ 3 mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous β 1 - and β 2 ARs, isoproterenol resulted in an increase in contractility in the TGβ 3 mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human β 3 AR agonist L-755,507 significantly increased contractility in the TGβ 3 mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGβ 3 mice was investigated in terms of β 3 AR–G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGβ 3 mice with L-755,507 resulted in a pertussis toxin–insensitive 1.33-fold increase in [ 35 S]GTPγS loading and a 1.6-fold increase in adenylyl cyclase activity. Conclusions Cardiac overexpression of human β 3 ARs results in positive inotropy only on stimulation with a β 3 AR agonist. Overexpressed β 3 ARs couple to G s and activate adenylyl cyclase on agonist stimulation.

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected β2-adrenergic receptors (β2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, β-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the β2AR. Receptor ubiquitination required β-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of β-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a β2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of β-arrestin in mediating the ubiquitination of the β2AR and indicate that ubiquitination of the receptor and of β-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β-adrenergic signaling defects including down-regulation of myocardial β-adrenergic receptors (β-ARs), functional β-AR uncoupling, and an up-regulation of the β-AR kinase (βARK1). Adenoviral-mediated gene transfer of the human β 2 -AR or an inhibitor of βARK1 to these failing myocytes led to the restoration of β-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of βARK1 activity in the heart.

Aldosterone is 1 of the various hormones with detrimental functions for the failing heart, whose circulating levels are elevated post-myocardial infarction (MI) and in patients with chronic heart failure (HF) [(1)][1]. We have recently discovered that angiotensin II, acting through its type 1