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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.

Solute carrier proteins (SLCs) are a superfamily of transmembrane transporters that control essential physiological functions such as nutrient uptake, ion transport, and cellular waste eli...

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.

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.

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.

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.

GABAB receptors (GABABR) play a critical role in GABAergic neurotransmission in the brain and are thought to be one of the most promising targets for the treatment of drug addiction. GABABR positive allosteric modulators (PAMs) have shown promise as potential anti-addictive therapies, as they lack the sedative and muscle relaxant properties of full GABAB receptor agonists such as baclofen. The present study was aimed at developing novel, selective, and potent GABABR PAMs with efficacy on abuse-related effects of nicotine. We synthetized ~100 analogs of BHF177, a GABABR PAM that has been shown to inhibit nicotine taking and seeking, and tested their activity in multiple cell-based functional assays. Among these compounds, KK-92A displayed superior PAM properties at the GABABR. Interestingly, our results revealed the existence of pathway-selective differential modulation of GABABR signaling by the structurally related GABABR allosteric modulators BHF177 and KK-92A. In vivo, similarly to BHF177, KK-92A inhibited intravenous nicotine self-administration under both fixed- and progressive-ratio schedules of reinforcement in rats. In contrast to BHF177, KK-92A had no effect on food self-administration. Furthermore, KK-92A decreased cue-induced nicotine-seeking behavior without affecting food seeking. These results indicate that KK-92A is a selective GABABR PAM with efficacy in inhibition of the primary reinforcing and incentive motivational effects of nicotine, and attenuation of nicotine seeking, further confirming that GABABR PAMs may be useful antismoking medications.

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.

G protein-coupled receptors (GPCRs) constitute the largest and most diverse protein family in the human genome with over 800 members identified to date. They play critical roles in numerous cellular and physiological processes, including cell proliferation, differentiation, neurotransmission, development and apoptosis. Consequently, aberrant receptor activity has been demonstrated in numerous disorders/diseases, and as a result GPCRs have become the most successful drug target class in pharmaceuticals treating a wide variety of indications such as pain, inflammation, neurobiological and metabolic disorders. Many independent studies have also demonstrated a key role for GPCRs in tumourigenesis, establishing their involvement in cancer initiation, progression, and metastasis. Given the growing appreciation of the role(s) that GPCRs play in cancer pathogenesis, it is surprising to note that very few GPCRs have been effectively exploited in pursuit of anti-cancer therapies. The present review provides a broad overview of the roles that various GPCRs play in cancer growth and development, highlighting the potential of pharmacologically modulating these receptors for the development of novel anti-cancer therapeutics.

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.

GABAThe present study was aimed at developing novel, selective, and potent GABAWe synthetized ~100 analogs of BHF177, a GABAThese results indicate that KK-92A is a selective GABA

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

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.