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The hydroxycarboxylic acid family of receptors (ENSFM00500000271913, nomenclature as agreed by the NC-IUPHAR Subcommittee on Hydroxycarboxylic acid receptors [36, 12]) respond to organic acids, including the endogenous hydroxy carboxylic acids 3-hydroxy butyric acid and L-lactic acid, as well as the lipid lowering agents nicotinic acid (niacin), acipimox and acifran [53, 60, 65]. These receptors were provisionally described as nicotinic acid receptors, although nicotinic acid shows submicromolar potency at HCA2 receptors only and is unlikely to be the natural ligand [60, 65].

PARP7 is a cellular stress-induced enzyme that adds mono-ADP-ribose groups to a variety of substrate proteins thereby regulating their function. One such substrate is the kinase TBK1 which regulates activity of cGAS-STING and RIG-I nucleic acid sensing pathways. Up-regulation of PARP7 expression in cancers applies a brake to cytosolic nucleic acid sensing and the Type I interferon response. This creates an immunosuppressive tumor microenvironment (TME) leading to faster tumor growth. In addition, inhibition of PARP7 has also been shown to directly arrest growth in a subset of cancer cells via the promotion of a senescence phenotype, inhibition of autophagy and regulation of metabolism. Through the use of structure-based drug design we describe the characterization of potent and selective inhibitors of PARP7. These molecules block viability of a subset of immortalized and primary cancer cells in 2D culture. We have observed potent cGAS-STING independent growth arrest in a number of cancer cell lines harboring a KRAS G12C mutation, and studies combining PARP7 inhibitors with commercially available KRAS G12C inhibitors yield additional in vitro efficacy. Recent studies have shown that inhibitors targeting oncogenic RAS pathway signaling can activate ULK1/2-mediated autophagy as an adaptive treatment resistance mechanism. Hence, our data support further studies investigating the use of PARP7 inhibitors in KRAS-driven cancers either as single agents or in combination with mutant KRAS inhibitors. Our molecules also stimulate robust induction of type I interferon (IFNβ) and downstream chemokines such as CXCL10 in a cGAS-STING pathway dependent manner in a range of cancer cells and 3D organoids. This induction is augmented by combining agonists of cGAS-STING and RIG-I pathways with PARP7 inhibitors. In addition, PARP7 inhibitors work in concert with exogenous DNA-damaging agents such as chemotherapeutics and X-ray radiation to induce the type I interferon response in cancer cells. Overall, these data highlight the opportunity to utilize PARP7 inhibitors in cancers where high genomic instability leads to aberrant cytosolic nucleic acid levels or in concert with exogenous DNA-damaging agents. Our PARP7 inhibitors exhibit highly desirable physico-chemical and in vitro ADME properties coupled with an exceptional in vitro safety profile which translates to best-in-class rodent PK. In a KRAS G12C lung cancer xenograft model (NCI-H1373), twice daily oral administration of an exemplar PARP7 inhibitor drives rapid and full tumor regression in a dose-dependent manner. The anti-tumor effects correlate with robust tumor PK/PD upon measurement of tumor IFNβ and CXCL10. In summary, we describe the characterization of a lead series of highly potent and selective PARP7 inhibitors which demonstrate excellent in vitro ADMET and in vivo PK properties leading to best-in-class anti-tumor efficacy in a KRAS-driven lung cancer xenograft model. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP7 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 486.

First-generation PARP1 inhibitors have provided significant therapeutic benefit to patients whose tumors exhibit homologous repair deficiencies, including BRCA mutations, however their use has been associated with hematological toxicities that have restricted their application, particularly in combination with standard-of-care chemotherapy. All four FDA-approved PARP1 inhibitors are largely non-selective for the closely related enzyme PARP2, inhibition of which has been shown to drive hematotoxicity. Hence, the development of second-generation molecules highly selective for PARP1 over PARP2 offers a significant opportunity to 1) dramatically enhance therapeutic index, 2) enable additional precision medicine/combination approaches with chemotherapy, radiotherapy, immunotherapy and targeted agents and 3) expand the addressable patient population to those whose tumors harbor additional DDR defects. Utilizing X-ray crystallography and structure-based drug design, we describe the characterization of a novel chemical series of highly selective PARP1 inhibitors. These molecules demonstrate high potency in multiple biochemical and cell-based assays, including viability and colony forming unit read-outs in BRCA-deficient cancer cells, whilst sparing isogenically-paired BRCA wild-type cells and non-transformed primary cell lines. The molecules are potent PARP1-DNA trappers and exhibit high affinity, high selectivity and prolonged residence time in biophysical surface plasmon resonance (SPR) binding assays. The molecules also demonstrate exceptionally high selectivity for PARP1 over PARP2, and across the mono and polyPARP family, using a cell-based NanoBRET target engagement assay. This chemical series generally exhibits highly desirable physico-chemical and in vitro ADME properties, coupled with an excellent in vitro safety profile, which translate to high oral bioavailability and low clearance in rodent PK studies. Lead molecules yield deep and durable anti-tumor efficacy in a BRCA1m MDA-MB-436 breast cancer xenograft model with responses continuing after cessation of treatment. Percent tumor regression and post-dose tumor control was superior to olaparib at 1/10th of the dose. Importantly, our molecules demonstrate prolonged tumor residence time and a markedly superior tumor:plasma ratio compared to competitor PARP1-selective inhibitors. Taken together, these data predict low therapeutic dosing with the potential to demonstrate improved efficacy and tolerability compared to marketed PARP inhibitors. In summary, we describe the characterization of novel potent and selective PARP1 inhibitors. These molecules demonstrate excellent in vitro ADMET and in vivo PK, coupled with profound anti-tumor efficacy and tumor-targeting properties in a genetically-defined mouse model, supporting their progression into clinical studies. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6172.

The G-protein-coupled receptors GPR81, GPR109A, and GPR109B share significant sequence homology and form a small group of receptors, each of which is encoded by clustered genes. In recent years, endogenous ligands for all three receptors have been described. These endogenous ligands have in common that they are hydroxy-carboxylic acid metabolites, and we therefore have proposed that this receptor family be named hydroxy-carboxylic acid (HCA) receptors. The HCA(1) receptor (GPR81) is activated by 2-hydroxy-propanoic acid (lactate), the HCA(2) receptor (GPR109A) is a receptor for the ketone body 3-hydroxy-butyric acid, and the HCA(3) receptor (GPR109B) is activated by the β-oxidation intermediate 3-hydroxy-octanoic acid. HCA(1) and HCA(2) receptors are found in most mammalian species, whereas the HCA(3) receptor is present only in higher primates. The three receptors have in common that they are expressed in adipocytes and are coupled to G(i)-type G-proteins mediating antilipolytic effects in fat cells. HCA(2) and HCA(3) receptors are also expressed in a variety of immune cells. HCA(2) is a receptor for the antidyslipidemic drug nicotinic acid (niacin) and related compounds, and there is an increasing number of synthetic ligands mainly targeted at HCA(2) and HCA(3) receptors. The aim of this article is to give an overview on the discovery and pharmacological characterization of HCAs, and to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature. We will also discuss open questions regarding this receptor family as well as their physiological role and therapeutic potential.

GPR55 is a G protein-coupled receptor activated by L-α-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB(1) and CB(2) receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl)aniline), which is approximately 60-fold selective for GPR55 (pEC(50) = 6.8) over GlyT1 (pIC(50) = 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.

Background: With the current obesity epidemic, the search for effective weight loss approaches is required. In the present study, changes in weight, body composition and cardiovascular (CV) risk in response to a low-fat, reduced-energy diet (LFRE), a low-carbohydrate/high-protein diet (LCHP), or a commercially available very low-calorie diet (LighterLife; LL) were assessed. Methods: One hundred and twenty obese patients (body mass index ≥35 kg/m2) underwent a screening period of 3 months on the LFRE. Those who lost >5% of their body weight were maintained on this approach for an additional 3 months, whereas those who lost >10% at this time were maintained for 1 year. Patients failing to achieve these targets were randomly allocated to either the LCHP (n = 38) or LL (n = 34) for a period of 9 months. Results: Significantly greater weight loss was seen for patients on the LL than the LCHP at 3 (mean (±SD) −11.6 ± 12.9 vs −2.8 ± 4.5 kg, respectively; P

Some growth hormone secretagogues act as agonists at the ghrelin receptor and have been described as "ago-allosteric" ligands because of an ability to also modulate the maximum efficacy and potency of ghrelin (Holst et al., 2005). In membranes prepared from cells coexpressing the human ghrelin receptor and the G protein Galpha(o1), N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4'-piperidin)-1'-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide (MK-677), growth hormone-releasing peptide 6 (GHRP-6), and the 2(R)-hydroxypropyl derivative of 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5-yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide (L-692,585) each functioned as direct agonists, and each displayed higher efficacy than ghrelin. The effect of multiple, fixed concentrations of each of these ligands on the function and concentration-dependence of ghrelin and the effect of multiple, fixed concentrations of ghrelin on the action of MK-677, GHRP-6, and L-692,585 was analyzed globally according to a modified version of an operational model of allosterism that accounts for allosteric modulation of affinity, efficacy, and allosteric agonism. Each of the data sets was best fit by a model of simple competition between a partial and a full agonist. Both positive and negative allosteric modulators are anticipated to alter the kinetics of binding of an orthosteric agonist. However, none of the proposed ago-allosteric regulators tested had any effect on the dissociation kinetics of (125)I-[His]-ghrelin, and GHRP-6 and MK-677 were able to fully displace (125)I-[His]-ghrelin from the receptor. At least in the system tested, each of the ligands acted in a simple competitive fashion with ghrelin as demonstrated by analysis according to a model whereby ghrelin is a partial agonist with respect to each of the synthetic agonists tested.

It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Cell-based assays have become the method of choice for compound screening in drug discovery. Such assays are used in high-throughput screening to identify molecules for lead optimisation and to support subsequent chemistry programmes in developing clinical candidates.In this article we describe the technologies and working practices that have been adopted in our laboratory and others to improve assay consistency and reduce the costs associated with cell-based screening.The key to the success of cell-based screening is the adoption of automated methods of cell culture or the use of cryopreserved cells to reduce biological variability, and the use of simple assay protocols and automation solutions to improve assay performance. In addition, we describe the adoption of several working practices common to the manufacturing industry to reduce variability in the screening process. We describe how the adoption of these technologies and working practices within an industrial environment allows the use of cell-based assays for the identification and optimisation of new clinical candidates.

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

Measurement of intracellular calcium release following agonist challenge within cells expressing the relevant membrane protein is a commonly used format to derive structure-activity relationship (SAR) data within a compound profiling assay. The Fluorometric Imaging Plate Reader (FLIPR) has become the gold standard for this purpose. FLIPR traditionally uses cells that are maintained in continuous culture for compound profiling of iterative chemistry campaigns. This supply dictates that assays can only be run on 4 of 5 weekdays, or alternative cell culture machinery is required such that plating can occur remotely at the weekend. The data reported here demonstrate that high-quality compound profiling data can be generated from the use of cryopreserved cells and that these cells can also be plated at various densities to generate equivalent data between 24 and 72 h postplating. Hence, the authors report a method that allows data generation throughout the week and without the requirement of highly automated cell culture or continuous culture. (Journal of Biomolecular Screening XXXX:xx-xx)

The human dopamine D(2L) receptor couples promiscuously to multiple members of the Galpha(i/o) subfamily. Despite the high homology of the D(2L) and D(3) receptors, the G protein coupling specificity of the human D(3) receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D(2L) and D(3) receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual Galpha(i)-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D(3) receptor to Galpha(o1). Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D(3) receptor when coupled to Galpha(o1) compared with the D(2L) receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D(2L) receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D(3)/D(2) receptor in which the equivalent region of the D(3) receptor was exchanged for this sequence. This chimera displayed D(3)-like affinity for [(3)H]spiperone and potency for agonists but gained D(2)-like ability to couple to each of Galpha(i1-3) as well as Galpha(o1).

Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

A range of ligands displayed agonism at the long isoform of the human dopamine D(2) receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxin-resistant mutants of individual Galpha(i)-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [(35)S]GTPgammaS onto each of Galpha(i1),Galpha(i2),Galpha(i3), and Galpha(o1). By contrast, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when Galpha(o1) was the target G protein but an antagonist/inverse agonist at Galpha(i1),Galpha(i2), and Galpha(i3). In ligand binding assays, dopamine identified both high- and low-affinity states at each of the dopamine D(2) receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D(2) receptor was fused to Galpha(i1),Galpha(i2), or Galpha(i3). However, it bound to distinct high- and low-affinity states of the D(2) receptor-Galpha(o1) fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D(2) receptor when expression of pertussis toxin-resistant forms of each of Galpha(i1), Galpha(i2), Galpha(i3), and Galpha(o1) was induced, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of Galpha(o1). p-Tyramine displayed a protean ligand profile similar to that of (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D(2) receptor and may explain in vivo actions of this ligand.

The enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1) catalyse oxidation of the essential amino acid tryptophan (Trp) leading to the formation of immunosuppressive kynurenine (Kyn) pathway metabolites that dampen the immune response in the tumour microenvironment. Both IDO1 and TDO have been shown to be up-regulated in a variety of cancers and blockade of their activity has been shown to stimulate the anti-tumour immune response in pre-clinical animal models. We have discovered multiple novel chemical series of both highly selective and dual-acting inhibitors of IDO1 and TDO. Herein we describe their in vitro and in vivo characterisation.

Many cancer cells rely on aerobic glycolysis for energy production and targeting of this pathway is a potential strategy to inhibit cancer cell growth. In this study, inhibition of five glycolysis pathway molecules (GLUT1, HKII, PFKFB3, PDHK1 and LDH) using 9 inhibitors (Phloretin, Quercetin, STF31, WZB117, 3PO, 3-bromopyruvate, Dichloroacetate, Oxamic acid, NHI-1) was investigated in panels of breast and ovarian cancer cell line models. All compounds tested blocked glycolysis as indicated by increased extracellular glucose and decreased lactate production and also increased apoptosis. Sensitivity to several inhibitors correlated with the proliferation rate of the cell lines. Seven compounds had IC50 values that were associated with each other consistent with a shared mechanism of action. A synergistic interaction was revealed between STF31 and Oxamic acid when combined with the antidiabetic drug metformin. Sensitivity to glycolysis inhibition was also examined under a range of O2 levels (21% O2, 7% O2, 2% O2 and 0.5% O2) and greater resistance to the inhibitors was found at low oxygen conditions (7% O2, 2% O2 and 0.5% O2) relative to 21% O2 conditions. These results indicate growth of breast and ovarian cancer cell lines is dependent on all the targets examined in the glycolytic pathway with increased sensitivity to the inhibitors under normoxic conditions.

1. N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulphonamide (GW627368X) is a novel, potent and selective competitive antagonist of prostanoid EP4 receptors with additional human TP receptor affinity. 2. At recombinant human prostanoid EP4 receptors expressed in HEK293 cells, GW627368X produced parallel rightward shifts of PGE2 concentration-effect (E/[A]) curves resulting in an affinity (pKb) estimate of 7.9 +/- 0.4 and a Schild slpoe not significantly different from unity. The affinity was independent of the agonist used. 4. In rings of phenylephrine precontracted piglet saphenous vein, GW627368X (30-300 nM) produced parallel rightward displacement of PGE2 E/[A] curves (pKb = 9.2 +/- 0.2; slope = 1). 4. GW627368X appears to bind to human prostanoid TP receptors but not the TP receptors of other species. In human washed platelets, GW627368X (10 microM) produced 100% inhibition of U-46619 (EC100)-induced aggregation (approximate pA2 approximately 7.0). However, in rings of rabbit and piglet saphenous vein and of guinea-pig aorta GW627368X (10 microM) did not displace U-46619 E/[A] curves indicating an affinity of < 5.0 for rabbit and guinea-pig prostanoid TP receptors. 5. In functional assays GW627368X is devoid of both agonism and antagonist affinity for prostanoid CRTH2, EP2, EP3, IP and FP receptors. At prostanoid EP1 receptors, GW627368X was an antagonist with a pA2 of 6.0, and at prostanoid IP receptors the compound increased the maximum effect of iloprost by 55%. At rabbit prostanoid EP2 receptors the pA2 of GW627368X was < 5.0. 6. In competition radioligand bioassays, GW627368X had affinity for human prostanoid EP4 and TP receptors (pKi = 7.0 +/- 0.2 (n = 10) and 6.8 (n = 2), respectively). Affinity for all other human prostanoid receptors was < 5.3. 7. GW627368X will be a valuable tool to explore the role of the prostanoid EP4 receptor in many physiological and pathological settings.

The neuropeptide neuromedin U (NmU) shows considerable structural conservation across species. Within the body, it is widely distributed and in mammals has been implicated in physiological roles, including the regulation of feeding, anxiety, pain, blood flow, and smooth muscle contraction. Human NmU-25 (hNmU-25) and other NmU analogs were recently identified as ligands for two human orphan G protein-coupled receptors, subsequently named hNmU-R1 and hNmU-R2. These receptors have approximately 50% amino acid homology, and, at least in mammalian species, NmU-R1 and NmU-R2 are expressed predominantly in the periphery and central nervous system, respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in human embryonic kidney 293 cells, particularly to define their G protein coupling and the activation and regulation of signal transduction pathways. We show that these receptors couple to both Galpha(q/11) and Galpha(i). Activation of either receptor type causes a pertussis toxin-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a pertussis toxin-sensitive inhibition of adenylyl cyclase with subnanomolar potency for each. Activation of phospholipase C is sustained, but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signaling by either recombinant receptors or those expressed endogenously in isolated smooth muscle cells from rat fundus. Using several strategies, we show this to be a consequence of essentially irreversible binding of hNmU-25 to its receptors and that this is followed by ligand internalization. Despite structural differences between receptors, there were no apparent differences in their activation, coupling, or regulation.

The completion of the human genome sequencing project has identified approximately 720 genes that belong to the G-protein coupled receptor (GPCR) superfamily. Approximately half of these genes are thought to encode sensory receptors. Of the remaining 360 receptors, the natural ligand has been identified for approximately 210 receptors, leaving 150 so-called orphan GPCRs with no known ligand or function. The identification of ligands active at orphan GPCRs has been achieved through the development of a number of experimental approaches, including the screening of putative small molecule and peptide ligands, reverse pharmacology, and the use of bioinformatics to predict candidate ligands. In this review, we discuss the methodologies developed for the identification of ligands at orphan GPCRs and include examples of their successful application.

Following identification of the human motilin receptor, we isolated the rabbit orthologue by PCR amplification and found this to be 85% identical to the open reading frame of the human receptor. The protein encoded was 84% identical to the human polypeptide. In HEK293T cells transfected with the rabbit receptor, motilin concentration-dependently increased intracellular calcium mobilisation (pEC50=9.25). After transfection with Go1alpha, motilin similarly stimulated [35S]GTPgammaS binding (pEC50=8.87). Using both systems, similar values were obtained with the human receptor, with rank-order potencies of motilin=[Nle13]-motilin>erythromycin; ghrelin was ineffective. In circular muscle preparations of rabbit gastric antrum, [Nle13]-motilin 0.1-30 nM concentration-dependently increased the amplitude of electrically-evoked, neuronally-mediated contractions (pEC50=8.3); higher concentrations increased the muscle tension (30-3000 nM). Both responses to [Nle13]-motilin faded rapidly during its continual presence. Rat or human ghrelin 0.01-10 microM were without activity. Erythromycin 30-3000 nM and 10 microM, respectively, increased neuronal activity and muscle tension in rabbit stomach. Unlike [Nle13]-motilin, the increase in neuronal activity did not fade during continual presence of submaximally-effective concentrations of erythromycin; some fade was observed at higher concentrations. We conclude that the pharmacology of the rabbit motilin receptor is similar to the human orthologue and, when expressed as a recombinant, comparable to the native receptor. However, in terms of their ability to increase neuronal activity in rabbit stomach, [Nle13]-motilin and erythromycin are distinguished by different response kinetics, reflecting different rates of ligand degradation and/or interaction with the receptor.

This article documents a Web-based program that aims to aid students’ understanding of research methods and prepare them for an examination question that has a similar style. Twenty sentences were presented at random from a larger pool and each had two missing words that the student had to match from the complete list of words. Students generally agreed that the sentences could not easily be guessed by using grammatical rules and that the puzzle program had helped them to understand the material and prepare for the examination. The program was ranked by students third of the eight support materials supplied to the students with a median score of 7.53 from a maximum of 10.

The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.

Several lines of evidence suggested that the first gamma-aminobutyric acid B receptor to be cloned required an additional factor for functional expression. GABA(B1) was retained within the endoplasmic reticulum and failed to couple to signal transduction pathways on stimulation with agonists. In radioligand binding experiments it was found that although the affinity of antagonists showed a close agreement between rat brain membranes and membranes expressing the cloned receptor, agonist ligands were significantly weaker at recombinant receptors. Using the C-terminal tail as bait, a yeast two-hybrid screen was run against a human brain cDNA library and identified a second receptor, GABA(B2), as a major interacting protein. This interaction was confirmed by coimmunoprecipitation as well as extensive colocalization studies. Coexpression of the two seven-transmembrane proteins generated a fully functional receptor, which was expressed at the cell surface confirming the importance of receptor heterodimerization for GABA(B) receptor activity.

G-protein coupled receptors (GPCRs) represent possibly the most important target class of proteins for drug discovery. Over 30% of clinically marketed drugs are active at this receptor family. These drugs exhibit their activity at

A program has been written in Visual Basic that helps the lecturer to construct an interactive crossword in JavaScript for use in a Web browser. Puzzles have been used to provide self-assessment material for student revision and attitudes were evaluated by use of a closed questionnaire. Students’ attitudes after using the program were generally positive and students agreed with the proposition that lecturers should be encouraged to use crossword puzzles to support learning.

Functional human GABAB(1a,2) and GABAB(1b,2) receptors have been stably expressed in mammalian CHO K1 cells. Detailed characterization of GABAB ligand binding at each of the receptors has been compared using [3H]-CGP54626A. In cell membranes fractions, [3H]-CGP54626A bound to a single site with a KD of 1.51±1.12 nM, Bmax of 2.02±0.17 pmoles mg protein−1 and 0.86±0.20 nM, Bmax of 5.19±0.57 pmoles mg protein−1 for GABAB(1a,2) and GABAB(1b,2) respectively. In competition binding assays the rank order was identical for both GABAB receptors. For known GABAB agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABAB antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 2-OH Saclofen ABPA. The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl2 from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABAB receptors. A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABAB receptors. In competition assays, the rank order was the same for both GABAB(1a,2) and GABAB(1b,2) and consistent with that seen with cell membrane fractions. These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABAB receptor are pharmacologically indistinguishable. British Journal of Pharmacology (2000) 131, 1766–1774; doi:10.1038/sj.bjp.0703755

The typical Scottish diet is high in meat, dairy products and confectionery and low in cereals, vegetables and fresh fruit, although evidence suggests that consumers are becoming more “health” conscious. Research indicates that more people are eating out nowadays and requesting “healthy” choices when doing so. This present study examined, by means of a questionnaire, chefs’ knowledge of nutrition and their ability to apply this to recipes. A total of 25 chefs completed the study. Most chefs had received training and had a positive attitude towards “healthy” eating. The changes chefs made to recipes were partly in keeping with current dietary guidelines, particularly for fat and energy. Further research is needed into whether chefs actually know and understand specific dietary guidelines or whether they lack the practical skills needed to prepare “healthy” dishes. It is recommended that college courses should teach chefs “healthy” eating theory reinforced by practical skills.

γ-Aminobutyric acid type B (GABA B ) receptors mediate the metabotropic actions of the inhibitory neurotransmitter GABA. These seven-transmembrane receptors are known to signal primarily through activation of G proteins to modulate the action of ion channels or second messengers. The functional GABA B receptor is made up of a heterodimer consisting of two subunits, GABA B -R1 and GABA B -R2, which interact via coiled-coil domains in their C-terminal tails. By using a yeast two-hybrid approach, we have identified direct interactions between the C-terminal tails of GABA B -R1 and GABA B -R2 with two related transcription factors, CREB2 (ATF4) and ATFx. In primary neuronal cultures as well in recombinant Chinese hamster ovary cells expressing GABA B receptors, CREB2 is localized within the cytoplasm as well as the nucleus. Activation of the GABA B receptor by the specific agonist baclofen leads to a marked translocation and accumulation of CREB2 from the cytoplasm into the nucleus. We demonstrate that receptor stimulation results in activation of transcription from a CREB2 responsive reporter gene. Such a signaling mechanism is unique among Family C G protein-coupled receptors and, in the case of the GABA B receptor and CREB2, may play a role in long-term changes in the nervous system.

A study was conducted to investigate the relationship between five individual factors and compliance with advice from a decision-support system. Rates of compliance were also compared for two different types of computer advice: specific and nonspecific. Results suggest that there is no significant difference in compliance rates between the two types of advice. However four of the individual factors (gender, trust in the advice, previous task performance, and self-confidence in the task) were significantly related to compliance with the nonspecific advice, while one factor (trust in the advice) was significantly related to compliance with the specific advice.

Summary Antiserum raised against squid ( Loligo forbesi ) rhodopsin cross-reacted with proteins from microsomal and plasma membranes isolated from Sorghum bicolor and Pisum sativum . In S. bicolor the main crossreacting protein (SbR) presented a molecular mass of 38 kDa. SbR appeared to be more abundant in mesocotyl microsomal membranes than in leaf or root membranes. SbR showed similarities to G-protein coupled receptors (GPCRs) in that it required a high detergent concentration for extraction and exhibited diffuse antibody staining on blots similar to that of squid rhodopsin. In P. sativum hypocotyl membranes the strongest immunological signal corresponded to a 35 kDa polypeptide (PsR) that was shown to be enriched in plasma membranes. A P. sativum glycoprotein bearing the same Mr and membrane association behaviour as PsR could be purified by Con A affinity chromatography and elution with α methyl manoside. The chromatographic fractions containing the glycoprotein were shown to possess high and specific GTPγS binding activity, indicating association to G-proteins. The glycoprotein showed weak cross-reaction to anti-rhodopsin serum. N-terminal sequencing of the glycoprotein did not show high homology to other proteins in the databases but indicated a likely amphipathic character.

Fusion proteins between the human A(1) adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G-protein alpha subunit G(i1)alpha (A1/Gi), and between the human A(1) adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly G(i1)alpha (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [(35)S]GTPgammaS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [(35)S]GTPgammaS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G-protein coupled receptor, GFP and a G-protein alpha subunit.

The GABA(B) receptor has been shown to consist of a heterodimer of two related 7-transmembrane receptors GABAB-R1 and GABA(B)-R2. These receptors share close homology to the Ca2+-sensing receptor and also to the metabotropic glutamate receptors, which have also been shown to respond to extracellular calcium. We show here that the GABA(B) receptor also has Ca2+ sensing properties. Ca2+ (0.001-1 mM) potentiated the GABA stimulation of [35S]GTPgammaS binding in membranes prepared from CHO cells stably expressing the GABA(B)-R1/R2 heterodimer. The GABA EC50 was reduced from 72 to 7.7 microM by addition of 1 mM Ca2+, with no change in the maximum response. A similar effect was observed in membranes from rat brain cortex. Ca2+ also potentiated GABA inhibition of forskolin-stimulated cAMP levels in the CHO cells and enhanced coupling to GIRK K+ channels in Xenopus oocytes. Other divalent cations were ineffective. The effects of Ca2+ were found to be agonist dependent with baclofen having a reduced sensitivity compared to GABA. Calcium appears to act allosterically to enhance GABA responses at the GABA(B) receptor, however, unlike the Ca2+-sensing receptor and some of the mGluR family, Ca2+ does not act as a ligand in its own right.

High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.

HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the α subunits of Gi1 (C351G), Gi2 ...

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.

Fusion proteins were constructed between the porcine alpha2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein Gi1alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha2A-adrenoceptor-wild type Gi1alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of Vmax and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha2A-adrenoceptor-Cys351 Gly Gi1alpha fusion protein was only 44'S, of that for the alpha2A-adrenoceptor-wild type Gi1alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.

Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the α subunit of the G protein Gi1 and the porcine α2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Giα. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the K m for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, V max measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated α2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Giα was greater than for the fusion protein-constrained C351G Gi1α. α2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated α2A-adrenoreceptor-C351G Gi1α fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Co-expression of the alpha2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the alpha subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1alpha generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1alpha rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) alpha2A-adrenoreceptor and either C351G or C3S/C351G Gi1alpha also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1alpha are cytosolic proteins. Expression of these as chimeric alpha2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.

Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1 alpha were expressed in COS-7 cells along with the porcine alpha 2A-adrenoceptor. G2A/C3S/C351G Gi1 alpha and G2A/C351G Gi1 alpha were largely cytosolic and failed to interact with the agonist-occupied alpha 2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1 alpha was almost entirely particulate and the alpha 2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTP gamma S which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1 alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha 2A-adrenoceptor. Coexpression of C3S/C351G Gi1 alpha and the alpha 2A-adrenoceptor along with beta 1 and gamma 2 subunits increased the P2 membrane complement of the alpha subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the alpha 2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1 alpha by the alpha 2A-adrenoceptor compared to C351G Gi1 alpha, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1 alpha and C3S/C351G Gi1 alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G Gi1 alpha nor G2A/C3S/C351G Gi1 alpha resulted in effective regulation of adenylyl cyclase activity.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

This paper describes four simulations of functions found in a commercial dietary analysis program that are designed to be used in education. These include FitDiets, which illustrates the principle of energy balance and “healthiness” of diets, and programs that present a nutritional quiz, a food label and information on dietetic products.

Cells of clones of rat 1 fibroblasts transfected to express the molecularly defined α, α, or α adrenoreceptors and prelabeled with myo-[3H]inositol were each shown to generate high levels of inositol phosphates when exposed to the α1 adrenoreceptor agonist phenylephrine. Maintained exposure of each of these cells to phenylephrine resulted in a large down-regulation of the receptors and also a marked down-regulation of cellular levels of both of the phosphoinositidase C-linked G-proteins Gqα and Gα. To examine the mechanism of phenylephrine-induced down-regulation of Gqα and Gα, pulse-chase S-amino acid labeling experiments were performed with each of the α, α, and α adrenoreceptor-expressing cell lines. The rate of degradation of Gα/Gqα, which was adequately modeled by a monoexponential with half-life between 33 and 40 h in each of the cell lines in the absence of agonist, was accelerated substantially (some 4-fold) in the presence of phenylephrine. By contrast, the rate of degradation of the G-protein Gi2α, which would not be anticipated to be activated by members of the α1 adrenoreceptor family, was unaltered by the presence of phenylephrine. Levels of mRNA encoding Gqα and Gα were not substantially altered by exposure of the cells to phenylephrine in any of the cell lines studied.

The enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1) catalyse oxidation of the essential amino acid tryptophan (Trp) leading to the formation of immunosuppressive kynurenine (Kyn) pathway metabolites that dampen the immune response in the tumor microenvironment. Both IDO1 and TDO have been shown to be up-regulated in a variety of cancers and blockade of their activity has been shown to stimulate the anti-tumour immune response in pre-clinical animal models. We have discovered and optimized multiple novel chemical series of both highly selective and dual-acting inhibitors of IDO1 and TDO. We describe the characterization of molecules that we are progressing into the clinic. We have characterized the IDO1 and TDO-selective inhibitors and dual-acting inhibitors which demonstrate nM potencies. In IFNγ-stimulated A172 glioblastoma cells expressing both enzymes, the dual-acting molecules fully inhibit Kyn production, whilst selective inhibitors yield only a partial response, demonstrating the utility of dual enzyme blockade in cancer cells. The compounds also relieve inhibition of T-cell proliferation in cancer cell conditioned media/T-cell co-culture assays and demonstrate highly favourable physico-chemical and pharmacokinetic properties. These properties translate to superior PK/PD effects following oral dosing in rodents with profound and sustained modulation of plasma Kyn levels, combined with an exceptional safety profile. In a mouse model of LPS-induced IDO1 activation, our IDO1 inhibitors fully ablate elevation of Kyn in both plasma and lung in a dose-dependent manner. Following oral administration, the IOmet IDO1 inhibitors provide excellent single-agent tumor growth control in PAN02 pancreatic adenocarcinoma syngeneic models. In addition, combining IOmet IDO1 inhibitors with standard-of-care chemotherapy (gemcitabine/abraxane) in the PAN02 model completely inhibits tumor growth. In summary, we describe the characterization of novel potent, selective and dual-acting IDO1 and TDO inhibitors. These candidate molecules demonstrate excellent rodent in vivo PK/PD, safety and anti-tumor profiles, supporting their progression into the clinic. Citation Format: Alan Wise, Barry E. McGuinness, Sarah C. Trewick, Thomas J. Brown, Phillip M. Cowley. Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5115.

Hepatocellular carcinoma (HCC) is an endemic disease globally, with a 5-year survival rate of 15%. Current treatment of late-stage HCC involves the use of chemotherapy and sorafenib, a multi-kinase inhibitor. Metabolic studies have indicated HCC has a high glycolysis rate, and is surrounded by a hypoglycemic microenvironment. Given the need for therapeutic options for HCC and its glycolytic nature, this study explored the activity of novel GLUT1 inhibitors in varied glucose concentrations. To assess target inhibition, [3H] deoxy-D-glucose (2DG) uptake assays were performed in HEK293 cells stably over-expressing GLUT1-4 individually. Six HCC cell lines were cultured in media with standard high glucose (HG:25mM) and low glucose/lactic acidosis (LGLA: 5mM glucose/20mM lactic acid) to mimic the hypoglycemic microenvironment. IC50s were assessed using the SRB assay, and other phenotypes using standard methods. Four novel GLUT1 inhibitors (IOM1-4) from the same chemical series were identified from diversity screening. The compounds exhibited differential potencies to GLUT proteins at the sub-μM level. The inhibitors reduced the uptake of 2DG, the extrusion of lactate, and increased apoptosis. In HCC cells, IC50s were lowest with IOM1 and 2 and highest with IOM4 (Table 1). HEP3B cells displayed exceptional sensitivity to the inhibitors. The ranking of potency according to the IC50s correlated with the 2DG uptake studies. There was no correlation between GLUT protein levels and IC50s. Significant differences in sensitivity to inhibitors were observed in cells cultured in HG and LGLA conditions. Cells cultured in LGLA had consistently lower glucose uptake compared those in HG conditions. In LGLA conditions, cells with low levels of reactive oxygen species (ROS) were more sensitive to GLUT1 inhibitors compared to cells with high ROS levels. In conclusion, GLUT1 inhibition can be influenced by microenvironment glucose levels and could be a strategy for HCC treatment. IC50 values (uM) for respective inhibitors in respective HCC cell linesCELL LINESInhibitorCultureC3AHEP3BHUH7PLCSKHEP1SNU449IOM1HG>5.00.8±0.040.3±0.06>5.01.4±0.451.7±0.38IOM1LGLA>5.00.2±0.134.0±1.20>5.00.1±0.021.6±0.28IOM2HG4.7±0.445.00.1±0.080.2±0.093.2±1.290.1±0.021.3±0.42IOM3HG>5.00.2±0.040.9±0.222.3±0.251.1±0.641.6±0.40IOM3LGLA>5.05.00.4±0.092.3±1.13IOM4HG>5.02.8±0.354.3±1.494.6±1.404.7±0.85>5.0IOM4LGLA>5.01.4±0.493.8±1.10>5.03.1±0.56>5.0 Citation Format: Bhaskar Bhattacharya, Sanamerjit S. Mann, Min Ji Han, Sarah HH Low, Gim Hwa Tan, Barry E. McGuinness, Sarah C. Trewick, Phillip M. Cowley, Alan Wise, Richie Soong. Identification and activity of novel GLUT1 inhibitors in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1283.