Science.gov

Sample records for allosteric modulators pams

  1. SAR studies on carboxylic acid series M(1) selective positive allosteric modulators (PAMs).

    PubMed

    Kuduk, Scott D; Beshore, Douglas C

    2014-01-01

    There is mounting evidence from preclinical and early proof-of-concept studies suggesting that selective modulation of the M1 muscarinic receptor is efficacious in cognitive models of Alzheimer's disease (AD). A number of nonselective M1 muscarinic agonists have previously shown positive effects on cognitive function in AD patients, but were limited due to cholinergic adverse events thought to be mediated by pan activation of the M2 to M5 sub-types. Thus, there is a need to identify selective activators of the M1 receptor to evaluate their potential in cognitive disorders. One strategy to confer selectivity for M1 is the identification of allosteric agonists or positive allosteric modulators, which would target an allosteric site on the M1 receptor rather than the highly conserved orthosteric acetylcholine binding site. BQCA has been identified as a highly selective carboxylic acid M1 PAM and this review focuses on an extensive lead optimization campaign undertaken on this compound.

  2. Chemical Lead Optimization of a pan Gq mAChR M1, M3, M5 Positive Allosteric Modulator (PAM) Lead. Part II. Development of potent and highly selective M1 PAM

    PubMed Central

    Bridges, Thomas M.; Kennedy, J. Phillip; Noetzel, Meredith J.; Breininger, Micah L.; Gentry, Patrick R.; Conn, P. Jeffrey

    2010-01-01

    This Letter describes a chemical lead optimization campaign directed at VU0119498, a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) with the goal of developing a selective M1 PAM. An iterative library synthesis approach delivered a potent (M1 EC50 = 830 nM) and highly selective M1 PAM (>30 μM vs. M2-M5). PMID:20156687

  3. Synthesis and SAR of novel, 4-(phenylsulfamoyl)phenylacetamide mGlu4 positive allosteric modulators (PAMs) identified by functional high-throughput screening (HTS).

    PubMed

    Engers, Darren W; Gentry, Patrick R; Williams, Richard; Bolinger, Julie D; Weaver, C David; Menon, Usha N; Conn, P Jeffrey; Lindsley, Craig W; Niswender, Colleen M; Hopkins, Corey R

    2010-09-01

    Herein we disclose the synthesis and SAR of a series of 4-(phenylsulfamoyl)phenylacetamide compounds as mGlu(4) positive allosteric modulators (PAMs) that were identified via a functional HTS. An iterative parallel approach to these compounds culminated in the discovery of VU0364439 (11) which represents the most potent (19.8 nM) mGlu(4) PAM reported to date.

  4. Discovery of dual positive allosteric modulators (PAMs) of the metabotropic glutamate 2 receptor and CysLT1 antagonists for treating migraine headache.

    PubMed

    Blanco, Maria-Jesus; Benesh, Dana R; Knobelsdorf, James A; Khilevich, Albert; Cortez, Guillermo S; Mokube, Fese; Aicher, Thomas D; Groendyke, Todd M; Marmsater, Fredrik P; Tang, Tony P; Johnson, Kirk W; Clemens-Smith, Amy; Muhlhauser, Mark A; Swanson, Steven; Catlow, John; Emkey, Renee; Johnson, Michael P; Schkeryantz, Jeffrey M

    2017-01-15

    Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu2 receptor. The hydroxyacetophenone scaffold is a second series of mGlu2 PAMs we have identified. This series of molecules are potent mGlu2 potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.

  5. Exploration of Allosteric Agonism Structure-Activity Relationships within an Acetylene Series of Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254)

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Chun, Aspen; Zhou, Ya; Gogliotti, Rocco D.; Nguyen, Elizabeth D.; Gregory, Karen J.; Vinson, Paige N.; Rook, Jerri M.; Gogi, Kiran K.; Xiang, Zixiu; Bridges, Thomas M.; Daniels, J. Scott; Jones, Carrie; Niswender, Colleen M.; Meiler, Jens; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs. PMID:24050755

  6. Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254).

    PubMed

    Turlington, Mark; Noetzel, Meredith J; Chun, Aspen; Zhou, Ya; Gogliotti, Rocco D; Nguyen, Elizabeth D; Gregory, Karen J; Vinson, Paige N; Rook, Jerri M; Gogi, Kiran K; Xiang, Zixiu; Bridges, Thomas M; Daniels, J Scott; Jones, Carrie; Niswender, Colleen M; Meiler, Jens; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

    2013-10-24

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.

  7. Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGluR5) attenuate microglial activation.

    PubMed

    Xue, Fengtian; Stoica, Bogdan A; Hanscom, Marie; Kabadi, Shruti V; Faden, Alan I

    2014-01-01

    Traumatic brain injury causes progressive neurodegeneration associated with chronic microglial activation. Recent studies show that neurodegeneration and neuroinflammation after traumatic brain injury can be inhibited as late as one month in animals by the activation of the metabotropic glutamate receptor 5 in microglia using (RS)-2-chloro-5- hydroxy-phenylglycine. However, the therapeutic potential of this agonist is limited due to its relatively weak potency and brain permeability. To address such concerns, we evaluated the anti-inflammatory activities of several positive allosteric modulators using various in vitro assays, and found that 3,3'-difluorobenzaldazine, 3-cyano-N-(1,3-diphenyl-1H-pyrazol- 5-yl)benzamide and 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide showed significantly improved potency which makes them potential lead compounds for further development of positive allosteric modulators for the treatment of traumatic brain injury.

  8. Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design.

    PubMed

    Volgraf, Matthew; Sellers, Benjamin D; Jiang, Yu; Wu, Guosheng; Ly, Cuong Q; Villemure, Elisia; Pastor, Richard M; Yuen, Po-wai; Lu, Aijun; Luo, Xifeng; Liu, Mingcui; Zhang, Shun; Sun, Liang; Fu, Yuhong; Lupardus, Patrick J; Wallweber, Heidi J A; Liederer, Bianca M; Deshmukh, Gauri; Plise, Emile; Tay, Suzanne; Reynen, Paul; Herrington, James; Gustafson, Amy; Liu, Yichin; Dirksen, Akim; Dietz, Matthias G A; Liu, Yanzhou; Wang, Tzu-Ming; Hanson, Jesse E; Hackos, David; Scearce-Levie, Kimberly; Schwarz, Jacob B

    2016-03-24

    The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

  9. Design and synthesis of systemically active metabotropic glutamate subtype-2 and -3 (mGlu2/3) receptor positive allosteric modulators (PAMs): pharmacological characterization and assessment in a rat model of cocaine dependence.

    PubMed

    Dhanya, Raveendra-Panickar; Sheffler, Douglas J; Dahl, Russell; Davis, Melinda; Lee, Pooi San; Yang, Li; Nickols, Hilary Highfield; Cho, Hyekyung P; Smith, Layton H; D'Souza, Manoranjan S; Conn, P Jeffrey; Der-Avakian, Andre; Markou, Athina; Cosford, Nicholas D P

    2014-05-22

    As part of our ongoing small-molecule metabotropic glutamate (mGlu) receptor positive allosteric modulator (PAM) research, we performed structure-activity relationship (SAR) studies around a series of group II mGlu PAMs. Initial analogues exhibited weak activity as mGlu2 receptor PAMs and no activity at mGlu3. Compound optimization led to the identification of potent mGlu2/3 selective PAMs with no in vitro activity at mGlu1,4-8 or 45 other CNS receptors. In vitro pharmacological characterization of representative compound 44 indicated agonist-PAM activity toward mGlu2 and PAM activity at mGlu3. The most potent mGlu2/3 PAMs were characterized in assays predictive of ADME/T and pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu2/3 PAMs. On the basis of its overall profile, compound 74 was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in rats after intraperitoneal administration. These mGlu2/3 receptor PAMs have significant potential as small molecule tools for investigating group II mGlu pharmacology.

  10. Development of a Highly Potent, Novel M5 Positive Allosteric Modulator (PAM) Demonstrating CNS Exposure: 1-((1H-Indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380)

    PubMed Central

    2015-01-01

    A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36). PMID:25147929

  11. Allosteric Modulation of Purine and Pyrimidine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Göblyös, Anikó; IJzerman, Adriaan P.

    2011-01-01

    Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action. PMID:21586360

  12. Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs).

    PubMed

    Engers, Darren W; Niswender, Colleen M; Weaver, C David; Jadhav, Satyawan; Menon, Usha N; Zamorano, Rocio; Conn, P Jeffrey; Lindsley, Craig W; Hopkins, Corey R

    2009-07-23

    We report the synthesis and evaluation of a series of heterobiaryl amides as positive allosteric modulators of mGluR4. Compounds 9b and 9c showed submicromolar potency at both human and rat mGluR4. In addition, both 9b and 9c were shown to be centrally penetrant in rats using nontoxic vehicles, a major advance for the mGluR4 field.

  13. Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.

    PubMed

    Noetzel, Meredith J; Rook, Jerri M; Vinson, Paige N; Cho, Hyekyung P; Days, Emily; Zhou, Y; Rodriguez, Alice L; Lavreysen, Hilde; Stauffer, Shaun R; Niswender, Colleen M; Xiang, Zixiu; Daniels, J Scott; Jones, Carrie K; Lindsley, Craig W; Weaver, C David; Conn, P Jeffrey

    2012-02-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

  14. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  15. Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

    PubMed

    Lundström, L; Bissantz, C; Beck, J; Dellenbach, M; Woltering, T J; Wichmann, J; Gatti, S

    2017-02-16

    The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist (3)[H]-LY354740 and mGlu2 PAM (3)[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of (3)[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of (3)[H]-LY354740 binding sites. (3)[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr(3.40) and Asn(5.46). Also of remark, in the described experimental conditions S731A (Ser(5.42)) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

  16. Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

    PubMed

    Lundström, L; Bissantz, C; Beck, J; Dellenbach, M; Woltering, T J; Wichmann, J; Gatti, S

    2016-12-01

    The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist (3)[H]-LY354740 and mGlu2 PAM (3)[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of (3)[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of (3)[H]-LY354740 binding sites. (3)[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr(3.40) and Asn(5.46). Also of remark, in the described experimental conditions S731A (Ser(5.42)) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

  17. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    PubMed Central

    Watterson, Lucas R.; Olive, M. Foster

    2013-01-01

    Positive allosteric modulators (PAMs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF) in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications. PMID:24380895

  18. Discovery of 8-Trifluoromethyl-3-cyclopropylmethyl-7-[(4-(2,4-difluorophenyl)-1-piperazinyl)methyl]-1,2,4-triazolo[4,3-a]pyridine (JNJ-46356479), a Selective and Orally Bioavailable mGlu2 Receptor Positive Allosteric Modulator (PAM).

    PubMed

    Cid, Jose María; Tresadern, Gary; Vega, Juan Antonio; de Lucas, Ana Isabel; Del Cerro, Alcira; Matesanz, Encarnación; Linares, María Lourdes; García, Aránzazu; Iturrino, Laura; Pérez-Benito, Laura; Macdonald, Gregor J; Oehlrich, Daniel; Lavreysen, Hilde; Peeters, Luc; Ceusters, Marc; Ahnaou, Abdellah; Drinkenburg, Wilhelmus; Mackie, Claire; Somers, Marijke; Trabanco, Andrés A

    2016-09-22

    Positive allosteric modulators of the metabotropic glutamate 2 receptor have generated great interest in the past decade. There is mounting evidence of their potential as therapeutic agents in the treatment of multiple central nervous system disorders. We have previously reported substantial efforts leading to potent and selective mGlu2 PAMs. However, finding compounds with the optimal combination of in vitro potency and good druglike properties has remained elusive, in part because of the hydrophobic nature of the allosteric binding site. Herein, we report on the lead optimization process to overcome the poor solubility inherent to the advanced lead 6. Initial prototypes already showed significant improvements in solubility while retaining good functional activity but displayed new liabilities associated with metabolism and hERG inhibition. Subsequent subtle modifications efficiently addressed those issues leading to the identification of compound 27 (JNJ-46356479). This new lead represents a more balanced profile that offers a significant improvement on the druglike attributes compared to previously reported leads.

  19. Enhancing NMDA Receptor Function: Recent Progress on Allosteric Modulators

    PubMed Central

    2017-01-01

    The N-methyl-D-aspartate receptors (NMDARs) are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer's disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer's disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs) of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs. PMID:28163934

  20. The therapeutic promise of positive allosteric modulation of nicotinic receptors

    PubMed Central

    Uteshev, Victor V.

    2014-01-01

    In the central nervous system, deficits in cholinergic neurotransmission correlate with decreased attention and cognitive impairment, while stimulation of neuronal nicotinic acetylcholine receptors improves attention, cognitive performance and neuronal resistance to injury as well as produces robust analgesic and anti-inflammatory effects. The rational basis for the therapeutic use of orthosteric agonists and positive allosteric modulators (PAMs) of nicotinic receptors arises from the finding that functional nicotinic receptors are ubiquitously expressed in neuronal and non-neuronal tissues including brain regions highly vulnerable to traumatic and ischemic types of injury (e.g., cortex and hippocampus). Moreover, functional nicotinic receptors do not vanish in age-, disease- and trauma-related neuropathologies, but their expression and/or activation levels decline in a subunit- and brain region-specific manner. Therefore, augmenting the endogenous cholinergic tone by nicotinic agents is possible and may offset neurological impairments associated with cholinergic hypofunction. Importantly, because neuronal damage elevates extracellular levels of choline (a selective agonist of α7 nicotinic acetylcholine receptors) near the site of injury, α7-PAM-based treatments may augment pathology-activated α7-dependent auto-therapies where and when they are most needed (i.e., in the penumbra, post-injury). Thus, the nicotinic-PAM-based treatments are expected to be highly efficacious with fewer side effects as compared to a more indiscriminate action of exogenous orthosteric agonists. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs. PMID:24530419

  1. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

    PubMed

    Ng, Herman J; Whittemore, Edward R; Tran, Minhtam B; Hogenkamp, Derk J; Broide, Ron S; Johnstone, Timothy B; Zheng, Lijun; Stevens, Karen E; Gee, Kelvin W

    2007-05-08

    Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.

  2. Allosteric Modulation of Alpha7 Nicotinic Receptors: Mechanistic Insight through Metadynamics and Essential Dynamics.

    PubMed

    Grazioso, Giovanni; Sgrignani, Jacopo; Capelli, Romina; Matera, Carlo; Dallanoce, Clelia; De Amici, Marco; Cavalli, Andrea

    2015-12-28

    Increasing attention has recently been devoted to allosteric modulators, as they can provide inherent advantages over classic receptor agonists. In the field of nicotinic receptors (nAChRs), the main advantage is that allosteric modulators can trigger pharmacological responses, limiting receptor desensitization. Most of the known allosteric ligands are "positive allosteric modulators" (PAMs), which increase both sensitivity to receptor agonists and current amplitude. Intriguingly, some allosteric modulators are also able to activate the α7 receptor (α7-nAChR) even in the absence of orthosteric agonists. These compounds have been named "ago-allosteric modulators" and GAT107 has been studied in depth because of its unique mechanism of action. We here investigate by molecular dynamics simulations, metadynamics, and essential dynamics the activation mechanism of α7-nAChR, in the presence of different nicotinic modulators. We determine the free energy profiles associated with the closed-to-open motion of the loop C, and we highlight mechanistic differences observed in the presence of different modulators. In particular, we demonstrate that GAT107 triggers conformational motions and cross-talk similar to those observed when the α7-nACh receptor is in complex with both an agonist and an allosteric modulator.

  3. Allosteric Modulation of Metabotropic Glutamate Receptors

    PubMed Central

    Sheffler, Douglas J.; Gregory, Karen J.; Rook, Jerri M.; Conn, P. Jeffrey

    2013-01-01

    The development of receptor subtype-selective ligands by targeting allosteric sites of G protein-coupled receptors (GPCRs) has proven highly successful in recent years. One GPCR family that has greatly benefited from this approach is the metabotropic glutamate receptors (mGlus). These family C GPCRs participate in the neuromodulatory actions of glutamate throughout the CNS, where they play a number of key roles in regulating synaptic transmission and neuronal excitability. A large number of mGlu subtype-selective allosteric modulators have been identified, the majority of which are thought to bind within the transmembrane regions of the receptor. These modulators can either enhance or inhibit mGlu functional responses and, together with mGlu knockout mice, have furthered the establishment of the physiologic roles of many mGlu subtypes. Numerous pharmacological and receptor mutagenesis studies have been aimed at providing a greater mechanistic understanding of the interaction of mGlu allosteric modulators with the receptor, which have revealed evidence for common allosteric binding sites across multiple mGlu subtypes and the presence for multiple allosteric sites within a single mGlu subtype. Recent data have also revealed that mGlu allosteric modulators can display functional selectivity toward particular signal transduction cascades downstream of an individual mGlu subtype. Studies continue to validate the therapeutic utility of mGlu allosteric modulators as a potential therapeutic approach for a number of disorders including anxiety, schizophrenia, Parkinson’s disease, and Fragile X syndrome. PMID:21907906

  4. Computational analysis of negative and positive allosteric modulator binding and function in metabotropic glutamate receptor 5 (in)activation.

    PubMed

    Dalton, James A R; Gómez-Santacana, Xavier; Llebaria, Amadeu; Giraldo, Jesús

    2014-05-27

    Metabotropic glutamate receptors (mGluRs) are high-profile G-protein coupled receptors drug targets because of their involvement in several neurological disease states, and mGluR5 in particular is a subtype whose controlled allosteric modulation, both positive and negative, can potentially be useful for the treatment of schizophrenia and relief of chronic pain, respectively. Here we model mGluR5 with a collection of positive and negative allosteric modulators (PAMs and NAMs) in both active and inactive receptor states, in a manner that is consistent with experimental information, using a specialized protocol that includes homology to increase docking accuracy, and receptor relaxation to generate an individual induced fit with each allosteric modulator. Results implicate two residues in particular for NAM and PAM function: NAM interaction with W785 for receptor inactivation, and NAM/PAM H-bonding with S809 for receptor (in)activation. Models suggest the orientation of the H-bond between allosteric modulator and S809, controlled by PAM/NAM chemistry, influences the position of TM7, which in turn influences the shape of the allosteric site, and potentially the receptor state. NAM-bound and PAM-bound mGluR5 models also reveal that although PAMs and NAMs bind in the same pocket and share similar binding modes, they have distinct effects on the conformation of the receptor. Our models, together with the identification of a possible activation mechanism, may be useful in the rational design of new allosteric modulators for mGluR5.

  5. Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships

    PubMed Central

    Gregory, Karen J.; Noetzel, Meredith J.; Rook, Jerri M.; Vinson, Paige N.; Stauffer, Shaun R.; Rodriguez, Alice L.; Emmitte, Kyle A.; Zhou, Ya; Chun, Aspen C.; Felts, Andrew S.; Chauder, Brian A.; Lindsley, Craig W.; Niswender, Colleen M.

    2012-01-01

    Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu5) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu5 to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu5-mediated Ca2+ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities. PMID:22863693

  6. Ligand-Based Discovery of a New Scaffold for Allosteric Modulation of the μ-Opioid Receptor.

    PubMed

    Bisignano, Paola; Burford, Neil T; Shang, Yi; Marlow, Brennica; Livingston, Kathryn E; Fenton, Abigail M; Rockwell, Kristin; Budenholzer, Lauren; Traynor, John R; Gerritz, Samuel W; Alt, Andrew; Filizola, Marta

    2015-09-28

    With the hope of discovering effective analgesics with fewer side effects, attention has recently shifted to allosteric modulators of the opioid receptors. In the past two years, the first chemotypes of positive or silent allosteric modulators (PAMs or SAMs, respectively) of μ- and δ-opioid receptor types have been reported in the literature. During a structure-guided lead optimization campaign with μ-PAMs BMS-986121 and BMS-986122 as starting compounds, we discovered a new chemotype that was confirmed to display μ-PAM or μ-SAM activity depending on the specific substitutions as assessed by endomorphin-1-stimulated β-arrestin2 recruitment assays in Chinese Hamster Ovary (CHO)-μ PathHunter cells. The most active μ-PAM of this series was analyzed further in competition binding and G-protein activation assays to understand its effects on ligand binding and to investigate the nature of its probe dependence.

  7. Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor.

    PubMed

    Burford, Neil T; Livingston, Kathryn E; Canals, Meritxell; Ryan, Molly R; Budenholzer, Lauren M L; Han, Ying; Shang, Yi; Herbst, John J; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Filizola, Marta; Bassoni, Daniel L; Wehrman, Tom S; Christopoulos, Arthur; Traynor, John R; Gerritz, Samuel W; Alt, Andrew

    2015-05-28

    Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulatorsPAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

  8. Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

    PubMed

    Croy, Carrie H; Schober, Douglas A; Xiao, Hongling; Quets, Anne; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(β,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved

  9. The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.

    PubMed

    Chatzidaki, Anna; D'Oyley, Jarryl M; Gill-Thind, JasKiran K; Sheppard, Tom D; Millar, Neil S

    2015-10-01

    Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties.

  10. Difluorocyclobutylacetylenes as positive allosteric modulators of mGluR5 with reduced bioactivation potential.

    PubMed

    Degnan, Andrew P; Maxwell, Darrell; Balakrishnan, Anand; Brown, Jeffrey M; Easton, Amy; Gulianello, Michael; Hanumegowda, Umesh; Hill-Drzewi, Melissa; Miller, Regina; Santone, Kenneth S; Senapati, Arun; Shields, Eric E; Sivarao, Digavalli V; Westphal, Ryan; Whiterock, Valerie J; Zhuo, Xiaoliang; Bronson, Joanne J; Macor, John E

    2016-12-15

    Schizophrenia is a serious illness that affects millions of patients and has been associated with N-methyl-d-aspartate receptor (NMDAR) hypofunction. It has been demonstrated that activation of metabotropic glutamate receptor 5 (mGluR5) enhances NMDA receptor function, suggesting the potential utility of mGluR5 positive allosteric modulators (PAMs) in the treatment of schizophrenia. Herein we describe the optimization of an mGluR5 PAM by replacement of a phenyl with aliphatic heterocycles and carbocycles as a strategy to reduce bioactivation in a biaryl acetylene chemotype. Replacement with a difluorocyclobutane followed by further optimization culminated in the identification of compound 32, a low fold shift PAM with reduced bioactivation potential. Compound 32 demonstrated favorable brain uptake and robust efficacy in mouse novel object recognition (NOR) at low doses.

  11. Metabotropic glutamate receptor 5 positive allosteric modulators are neuroprotective in a mouse model of Huntington's disease

    PubMed Central

    Doria, JG; Silva, FR; Souza, JM; Vieira, LB; Carvalho, TG; Reis, HJ; Pereira, GS; Dobransky, T; Ribeiro, FM

    2013-01-01

    Background and Purpose Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. We have previously demonstrated that the cell signalling of the metabotropic glutamate receptor 5 (mGluR5) is altered in a mouse model of HD. Although mGluR5-dependent protective pathways are more activated in HD neurons, intracellular Ca2+ release is also more pronounced, which could contribute to excitotoxicity. In the present study, we aim to investigate whether mGluR5 positive allosteric modulators (PAMs) could activate protective pathways without triggering high levels of Ca2+ release and be neuroprotective in HD. Experimental Approach We performed a neuronal cell death assay to determine which drugs are neuroprotective, Western blot and Ca2+ release experiments to investigate the molecular mechanisms involved in this neuroprotection, and object recognition task to determine whether the tested drugs could ameliorate HD memory deficit. Key Results We find that mGluR5 PAMs can protect striatal neurons from the excitotoxic neuronal cell death promoted by elevated concentrations of glutamate and NMDA. mGluR5 PAMs are capable of activating Akt without triggering increased intracellular Ca2+ concentration ([Ca2+]i); and Akt blockage leads to loss of PAM-mediated neuroprotection. Importantly, PAMs' potential as drugs that may be used to treat neurodegenerative diseases is highlighted by the neuroprotection exerted by mGluR5 PAMs on striatal neurons from a mouse model of HD, BACHD. Moreover, mGluR5 PAMs can activate neuroprotective pathways more robustly in BACHD mice and ameliorate HD memory deficit. Conclusions and Implications mGluR5 PAMs are potential drugs that may be used to treat neurodegenerative diseases, especially HD. PMID:23489026

  12. 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidines as mGluR2 positive allosteric modulators for the treatment of psychosis.

    PubMed

    Zhang, Lei; Brodney, Michael A; Candler, John; Doran, Angela C; Duplantier, Allen J; Efremov, Ivan V; Evrard, Edel; Kraus, Kenneth; Ganong, Alan H; Haas, Jessica A; Hanks, Ashley N; Jenza, Keith; Lazzaro, John T; Maklad, Noha; McCarthy, Sheryl A; Qian, Weimin; Rogers, Bruce N; Rottas, Melinda D; Schmidt, Christopher J; Siuciak, Judith A; Tingley, F David; Zhang, Andy Q

    2011-03-24

    A novel series of mGluR2 positive allosteric modulators (PAMs), 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidines, is herein disclosed. Structure-activity relationship studies led to potent, selective mGluR2 PAMs with excellent pharmacokinetic profiles. A representative lead compound (+)-17e demonstrated dose-dependent inhibition of methamphetamine-induced hyperactivity and mescaline-induced scratching in mice, providing support for potential efficacy in treating psychosis.

  13. 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

    PubMed Central

    Newman, Amy S; Batis, Nikolaos; Grafton, Gillian; Caputo, Francesca; Brady, Catherine A; Lambert, Jeremy J; Peters, John A; Gordon, John; Brain, Keith L; Powell, Andrew D; Barnes, Nicholas M

    2013-01-01

    Background and Purpose The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. Experimental Approach 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor. Linked Articles Recent reviews on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2

  14. New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2): identification and synthesis of N-propyl-8-chloro-6-substituted isoquinolones.

    PubMed

    Trabanco, Andrés A; Duvey, Guillaume; Cid, José María; Macdonald, Gregor J; Cluzeau, Philippe; Nhem, Vanthea; Furnari, Rocco; Behaj, Nadia; Poulain, Géraldine; Finn, Terry; Lavreysen, Hilde; Poli, Sonia; Raux, Alexandre; Thollon, Yves; Poirier, Nicolas; D'Addona, David; Andrés, José Ignacio; Lutjens, Robert; Le Poul, Emmanuel; Imogai, Hassan; Rocher, Jean-Philippe

    2011-02-01

    A series of N-propyl-8-chloro-6-substituted isoquinolones was identified as positive allosteric modulators of metabotropic glutamate receptor 2 (mGluR2 PAM) via high throughput screening (HTS). The subsequent synthesis and initial SAR exploration that led to the identification of compound 28 is described.

  15. Triazolopyridine ethers as potent, orally active mGlu2 positive allosteric modulators for treating schizophrenia.

    PubMed

    Higgins, Mendi A; Marcin, Lawrence R; Christopher Zusi, F; Gentles, Robert; Ding, Min; Pearce, Bradley C; Easton, Amy; Kostich, Walter A; Seager, Matthew A; Bourin, Clotilde; Bristow, Linda J; Johnson, Kim A; Miller, Regina; Hogan, John; Whiterock, Valerie; Gulianello, Michael; Ferrante, Meredith; Huang, Yanling; Hendricson, Adam; Alt, Andrew; Macor, John E; Bronson, Joanne J

    2017-01-15

    Triazolopyridine ethers with mGlu2 positive allosteric modulator (PAM) activity are disclosed. The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided. The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule BMT-133218 ((+)-7m). After oral administration at 10mg/kg, BMT-133218 demonstrated full reversal of PCP-stimulated locomotor activity and prevented MK-801-induced working memory deficits in separate mouse models. Also, reversal of impairments in executive function were observed in rat set-shifting studies at 3 and 10mg/kg (p.o.). Extensive plasma protein binding as the result of high lipophilicity likely limited activity at lower doses. Optimized triazolopyridine ethers offer utility as mGlu2 PAMs for the treatment of schizophrenia and merit further preclinical investigation.

  16. 3-Benzyl-1,3-oxazolidin-2-ones as mGluR2 positive allosteric modulators: Hit-to lead and lead optimization.

    PubMed

    Duplantier, Allen J; Efremov, Ivan; Candler, John; Doran, Angela C; Ganong, Alan H; Haas, Jessica A; Hanks, Ashley N; Kraus, Kenneth G; Lazzaro, John T; Lu, Jiemin; Maklad, Noha; McCarthy, Sheryl A; O'Sullivan, Theresa J; Rogers, Bruce N; Siuciak, Judith A; Spracklin, Douglas K; Zhang, Lei

    2009-05-01

    The discovery, synthesis and SAR of a novel series of 3-benzyl-1,3-oxazolidin-2-ones as positive allosteric modulators (PAMs) of mGluR2 is described. Expedient hit-to-lead work on a single HTS hit led to the identification of a ligand-efficient and structurally attractive series of mGluR2 PAMs. Human microsomal clearance and suboptimal physicochemical properties of the initial lead were improved to give potent, metabolically stable and orally available mGluR2 PAMs.

  17. GABAB receptors as a therapeutic strategy in substance use disorders: focus on positive allosteric modulators.

    PubMed

    Filip, Małgorzata; Frankowska, Małgorzata; Sadakierska-Chudy, Anna; Suder, Agata; Szumiec, Lukasz; Mierzejewski, Paweł; Bienkowski, Przemyslaw; Przegaliński, Edmund; Cryan, John F

    2015-01-01

    γ-Aminobutyric acid B (GABAB) receptors and their ligands are postulated as potential therapeutic targets for the treatment of several brain disorders, including drug dependence. Over the past fifteen years positive allosteric modulators (PAMs) have emerged that enhance the effects of GABA at GABAB receptors and which may have therapeutic effects similar to those of agonists but with superior side-effect profiles. This review summarizes current preclinical evidence supporting a role of GABAB receptor PAMs in drug addiction in several paradigms with relevance to reward processes and drug abuse liability. Extensive behavioral research in recent years has indicated that PAMs of GABAB receptors may have a therapeutic efficacy in cocaine, nicotine, amphetamine and alcohol dependence. The magnitude of the effects observed are similar to that of the clinically approved drug baclofen, an agonist at GABAB receptors. Moreover, given that anxiolytic effects are also reported with such ligands they may also benefit in mitigating the withdrawal from drugs of abuse. In summary, a wealth of data now supports the benefits of GABAB receptor PAMs and clinical validation is now warranted.

  18. The Antinociceptive and Antiinflammatory Properties of 3-furan-2-yl-N-p-tolyl-acrylamide, a Positive Allosteric Modulator of α7 Nicotinic Acetylcholine Receptors in Mice

    PubMed Central

    Bagdas, Deniz; Targowska-Duda, Katarzyna M.; López, Jhon J.; Perez, Edwin G.; Arias, Hugo R.; Damaj, M. Imad

    2016-01-01

    BACKGROUND Positive allosteric modulators (PAMs) facilitate endogenous neurotransmission and/or enhance the efficacy of agonists without directly acting on the orthosteric binding sites. In this regard, selective α7 nicotinic acetylcholine receptor type II PAMs display antinociceptive activity in rodent chronic inflammatory and neuropathic pain models. This study investigates whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a new putative α7-selective type II PAM, attenuates experimental inflammatory and neuropathic pains in mice. METHODS We tested the activity of PAM-2 after intraperitoneal administration in 3 pain assays: the carrageenan-induced inflammatory pain, the complete Freund adjuvant induced inflammatory pain, and the chronic constriction injury–induced neuropathic pain in mice. We also tested whether PAM-2 enhanced the effects of the selective α7 agonist choline in the mouse carrageenan test given intrathecally. Because the experience of pain has both sensory and affective dimensions, we also evaluated the effects of PAM-2 on acetic acid–induced aversion by using the conditioned place aversion test. RESULTS We observed that systemic administration of PAM-2 significantly reversed mechanical allodynia and thermal hyperalgesia in inflammatory and neuropathic pain models in a dose- and time-dependent manner without motor impairment. In addition, by attenuating the paw edema in inflammatory models, PAM-2 showed antiinflammatory properties. The antinociceptive effect of PAM-2 was inhibited by the selective competitive antagonist methyllycaconitine, indicating that the effect is mediated by α7 nicotinic acetylcholine receptors. Furthermore, PAM-2 enhanced the antiallodynic and antiinflammatory effects of choline, a selective α7 agonist, in the mouse carrageenan test. PAM-2 was also effective in reducing acetic acid induced aversion in the conditioned place aversion assay. CONCLUSIONS These findings suggest that the administration of PAM-2, a new α7

  19. Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.

    PubMed

    Grønlien, Jens Halvard; Håkerud, Monika; Ween, Hilde; Thorin-Hagene, Kirsten; Briggs, Clark A; Gopalakrishnan, Murali; Malysz, John

    2007-09-01

    Selective modulation of alpha7 nicotinic acetylcholine receptors (nAChRs) is thought to regulate processes impaired in schizophrenia, Alzheimer's disease, and other dementias. One approach to target alpha7 nAChRs is by positive allosteric modulation. Structurally diverse compounds, including PNU-120596, 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), and 5-hydroxyindole (5-HI) have been identified as positive allosteric modulators (PAMs), but their receptor interactions and pharmacological profiles remain to be fully elucidated. In this study, we investigated interactions of these compounds at human alpha7 nAChRs, expressed in Xenopus laevis oocytes, along with genistein, a tyrosine kinase inhibitor. Genistein was found to function as a PAM. Two types of PAM profiles were observed. 5-HI and genistein predominantly affected the apparent peak current (type I) whereas PNU-120596 and TQS increased the apparent peak current and evoked a distinct weakly decaying current (type II). Concentration-responses to agonists [ACh, 3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine dihydrochloride (GTS-21), and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)] were potentiated by both types, although type II PAMs had greater effects. When applied after alpha7 nAChRs were desensitized, type II, but not type I, PAMs could reactivate alpha7 currents. Both types of PAMs also increased the ACh-evoked alpha7 window currents, with type II PAMs generally showing larger potentiation. None of the PAMs tested increased nicotine-evoked Ca(2+) transients in human embryonic kidney 293 cells expressing human alpha4beta2 or alpha3beta4 nAChRs, although some inhibition was noted for 5-HI, genistein, and TQS. In summary, our studies reveal two distinct alpha7 PAM profiles, which could offer unique opportunities for modulating alpha7 nAChRs in vivo and in the development of novel

  20. Positive allosteric modulators of the μ-opioid receptor: a novel approach for future pain medications

    PubMed Central

    Burford, N T; Traynor, J R; Alt, A

    2015-01-01

    Morphine and other agonists of the μ-opioid receptor are used clinically for acute and chronic pain relief and are considered to be the gold standard for pain medication. However, these opioids also have significant side effects, which are also mediated via activation of the μ-opioid receptor. Since the latter half of the twentieth century, researchers have sought to tease apart the mechanisms underlying analgesia, tolerance and dependence, with the hope of designing drugs with fewer side effects. These efforts have revolved around the design of orthosteric agonists with differing pharmacokinetic properties and/or selectivity profiles for the different opioid receptor types. Recently, μ-opioid receptor-positive allosteric modulators (μ-PAMs) were identified, which bind to a (allosteric) site on the μ-opioid receptor separate from the orthosteric site that binds an endogenous agonist. These allosteric modulators have little or no detectable functional activity when bound to the receptor in the absence of orthosteric agonist, but can potentiate the activity of bound orthosteric agonist, seen as an increase in apparent potency and/or efficacy of the orthosteric agonist. In this review, we describe the potential advantages that a μ-PAM approach might bring to the design of novel therapeutics for pain that may lack the side effects currently associated with opioid therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24460691

  1. Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

    PubMed

    Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

    2014-06-05

    A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.

  2. Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators.

    PubMed

    Yu, Henry N; Richardson, Thomas E; Nataraja, Selva; Fischer, David J; Sriraman, Venkataraman; Jiang, Xuliang; Bharathi, Pandi; Foglesong, Robert J; Haxell, Thomas F N; Heasley, Brian H; Jenks, Mathew; Li, Jane; Dugas, Melanie S; Collis, Regina; Tian, Hui; Palmer, Stephen; Goutopoulos, Andreas

    2014-05-01

    Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

  3. THE ANTIPSYCHOTIC POTENTIAL OF MUSCARINIC ALLOSTERIC MODULATION

    PubMed Central

    Bridges, Thomas M.; LeBois, Evan P.; Hopkins, Corey R.; Wood, Michael R.; Jones, Carrie K.; Conn, P. Jeffrey; Lindsley, Craig W.

    2016-01-01

    SUMMARY The cholinergic hypothesis of schizophrenia emerged over 50 years ago based on clinical observations with both anticholinergics and pan-muscarinic agonists. Not until the 1990s did the cholinergic hypothesis of schizophrenia receive renewed enthusiasm based on clinical data with xanomeline, a muscarinic acetylcholine receptor M1/M4-preferring orthosteric agonist. In a clinical trial with Alzheimer’s patients, xanomeline not only improved cognitive performance, but also reduced psychotic behaviors. This encouraging data spurred a second clinical trial in schizophrenic patients, wherein xanomeline significantly improved the positive, negative and cognitive symptom clusters. However, the question remained: Was the antipsychotic efficacy due to activation of M1, M4 or both M1/M4? Classical orthosteric ligands lacked the muscarinic receptor subtype selectivity required to address this key question. More recently, functional assays have allowed for the discovery of ligands that bind at allosteric sites, binding sites distinct from the orthosteric (acetylcholine) site, which are structurally less conserved and thereby afford high levels of receptor subtype selectivity. Recently, allosteric ligands, with unprecedented selectivity for either M1 or M4, have been discovered and have demonstrated comparable efficacy to xanomeline in preclinical antipsychotic and cognition models. These data suggest that selective allosteric activation of either M1 or M4 has antipsychotic potential through distinct, yet complimentary mechanisms. PMID:20520852

  4. Allosteric Modulators for the Treatment of Schizophrenia: Targeting Glutamatergic Networks

    PubMed Central

    Menniti, Frank S.; Lindsley, Craig W.; Conn, P. Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A.

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved. PMID:23409764

  5. Allosteric modulation of GABA(A) receptor subtypes:effects on visual recognition and visuospatial working memory in rhesus monkeys [corrected].

    PubMed

    Soto, Paul L; Ator, Nancy A; Rallapalli, Sundari K; Biawat, Poonam; Clayton, Terry; Cook, James M; Weed, Michael R

    2013-10-01

    Non-selective positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) are known to impair anterograde memory. The role of the various GABAAR subtypes in the memory-impairing effects of non-selective GABAAR PAMs has not been fully elucidated. The current study assessed, in rhesus monkeys, effects of modulation of α1, α2/3, and α5GABAARs on visual recognition and spatial working memory using delayed matching-to-sample (DMTS) and self-ordered spatial search (SOSS) procedures, respectively. The DMTS procedure (n=8) involved selecting a previously presented 'sample' image from a set of multiple images presented after a delay. The SOSS procedure (n=6) involved touching a number of boxes without repeats. The non-selective GABAAR PAM triazolam and the α1GABAA preferential PAMS zolpidem and zaleplon reduced accuracy in both procedures, whereas the α5GABAA preferential PAMs SH-053-2'F-R-CH3 and SH-053-2'F-S-CH3, and the α2/3GABAA preferential PAM TPA023B were without effects on accuracy or trial completion. The low-efficacy α5GABAAR negative allosteric modulator (NAM) PWZ-029 slightly increased only DMTS accuracy, whereas the high-efficacy α5GABAAR NAMs RY-23 and RY-24 did not affect accuracy under either procedure. Finally, the slopes of the accuracy dose-effect curves for triazolam, zolpidem, and zaleplon increased with box number in the SOSS procedure, but were equivalent across DMTS delays. The present results suggest that (1) α1GABAARs, compared with α2/3 and α5GABAARs, are primarily involved in the impairment, by non-selective GABAAR PAMs, of visual recognition and visuospatial working memory in nonhuman primates; and (2) relative cognitive impairment produced by positive modulation of GABAARs increases with number of locations to be remembered, but not with the delay for remembering.

  6. Positive allosteric modulation of alpha 7 nicotinic acetylcholine receptors enhances recognition memory and cognitive flexibility in rats.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Potasiewicz, Agnieszka; Popik, Piotr

    2015-08-01

    A wide body of preclinical and clinical data suggests that alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) may represent useful targets for cognitive improvement in schizophrenia and Alzheimer׳s disease. A promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs due to their several advantages over the direct agonists. Nevertheless, the behavioural effects of this class of compounds, particularly with regard to higher-order cognitive functions, have not been broadly characterised. The aim of the present study was to evaluate the procognitive efficacies of type I and type II α7-nAChRs PAMs, N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) in the novel object recognition task (NORT), attentional set-shifting task (ASST) and five-choice serial reaction time task (5-CSRTT) in rats. Additionally, the effects of galantamine, an acetylcholinesterase inhibitor that also allosterically modulates nAChRs, were assessed. We report that CCMI (0.3-3mg/kg), PNU-120596 (0.3-3mg/kg) and galantamine (1-3mg/kg) attenuated the delay-induced impairment in NORT performance and facilitated cognitive flexibility in the ASST. Methyllycaconitine (3mg/kg) blocked the actions of CCMI, PNU-120596 and galantamine in the NORT and ASST, suggesting that the procognitive effects of these compounds are α7-nAChRs-dependent. However, none of the compounds tested affected the rats' attentional performance in the 5-CSRTT. The present findings confirm and extend the observations indicating that the positive allosteric modulation of α7-nAChRs enhances recognition memory and cognitive flexibility in preclinical tasks. Therefore, the present study supports the utility of α7-nAChRs PAMs as a potential cognitive enhancing therapy.

  7. Positive allosteric modulators of alpha 7 nicotinic acetylcholine receptors reverse ketamine-induced schizophrenia-like deficits in rats.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Hołuj, Małgorzata; Potasiewicz, Agnieszka; Popik, Piotr

    2016-02-01

    Alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) have generated great interest as targets of new pharmacological treatments for cognitive dysfunction in schizophrenia. One promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs, which demonstrate several advantages over direct agonists. Nevertheless, the efficacy of these newly introduced α7-nAChR agents has not been extensively characterised in animal models of schizophrenia. The aim of the present study was to evaluate the efficacy of type I and II PAMs, N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) and N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI), respectively, and galantamine, an acetylcholinesterase inhibitor (AChE) that also allosterically modulates nAChRs, against ketamine-induced cognitive deficits and social withdrawal in rats. The orthosteric α7-nAChR agonist octahydro-2-methyl-5-(6-phenyl-3-pyridazinyl)-pyrrolo[3,4-c]pyrrole (A-582941) was used as a positive control. Additionally, the antipsychotic activities of the tested compounds were assessed using the conditioned avoidance response (CAR) test. PNU-120596, CCMI, galantamine and A-582941 reversed ketamine-induced cognitive inflexibility, as assessed in the attentional set-shifting task (ASST). The tested compounds were also effective against ketamine-induced impairment in the novel object recognition task (NORT). PNU-120596, CCMI, and A-582941 ameliorated ketamine-induced social interaction deficits, whereas galantamine was ineffective. Moreover, all tested compounds selectively suppressed the CAR. The positive allosteric modulation of α7-nAChRs demonstrates preclinical efficacy not only against schizophrenia-like cognition impairments but also positive and negative symptoms. Therefore, the use of α7-nAChR PAMs as a potential treatment strategy in schizophrenia is supported.

  8. Biotransformation of a novel positive allosteric modulator of metabotropic glutamate receptor subtype 5 contributes to seizure-like adverse events in rats involving a receptor agonism-dependent mechanism.

    PubMed

    Bridges, Thomas M; Rook, Jerri M; Noetzel, Meredith J; Morrison, Ryan D; Zhou, Ya; Gogliotti, Rocco D; Vinson, Paige N; Xiang, Zixiu; Jones, Carrie K; Niswender, Colleen M; Lindsley, Craig W; Stauffer, Shaun R; Conn, P Jeffrey; Daniels, J Scott

    2013-09-01

    Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system disorders, including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 were discovered to exhibit in vivo efficacy in rodent models of psychosis, including PAMs possessing varying degrees of agonist activity (ago-PAMs), as well as PAMs devoid of agonist activity. However, previous studies revealed that ago-PAMs can induce seizure activity and behavioral convulsions, whereas pure mGlu5 PAMs do not induce these adverse effects. We recently identified a potent and selective mGlu5 PAM, VU0403602, that was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by coadministration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine. Consistent with potential adverse effects induced by ago-PAMs, we found that VU0403602 had significant allosteric agonist activity. Interestingly, inhibition of VU0403602 metabolism in vivo by a pan cytochrome P450 (P450) inactivator completely protected rats from induction of seizures. P450-mediated biotransformation of VU0403602 was discovered to produce another potent ago-PAM metabolite-ligand (M1) of mGlu5. Electrophysiological studies in rat hippocampal slices confirmed agonist activity of both M1 and VU0403602 and revealed that M1 can induce epileptiform activity in a manner consistent with its proconvulsant behavioral effects. Furthermore, unbound brain exposure of M1 was similar to that of the parent compound, VU0403602. These findings indicate that biotransformation of mGlu5 PAMs to active metabolite-ligands may contribute to the epileptogenesis observed after in vivo administration of this class of allosteric receptor modulators.

  9. mGluR5 positive allosteric modulators facilitate both hippocampal LTP and LTD and enhance spatial learning.

    PubMed

    Ayala, Jennifer E; Chen, Yelin; Banko, Jessica L; Sheffler, Douglas J; Williams, Richard; Telk, Alexandra N; Watson, Noreen L; Xiang, Zixiu; Zhang, Yongqin; Jones, Paulianda J; Lindsley, Craig W; Olive, M Foster; Conn, P Jeffrey

    2009-08-01

    Highly selective positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have emerged as a potential approach to treat positive symptoms associated with schizophrenia. mGluR5 plays an important role in both long-term potentiation (LTP) and long-term depression (LTD), suggesting that mGluR5 PAMs may also have utility in improving impaired cognitive function. However, if mGluR5 PAMs shift the balance of LTP and LTD or induce a state in which afferent activity induces lasting changes in synaptic function that are not appropriate for a given pattern of activity, this could disrupt rather than enhance cognitive function. We determined the effect of selective mGluR5 PAMs on the induction of LTP and LTD at the Schaffer collateral-CA1 synapse in the hippocampus. mGluR5-selective PAMs significantly enhanced threshold theta-burst stimulation (TBS)-induced LTP. In addition, mGluR5 PAMs enhanced both DHPG-induced LTD and LTD induced by the delivery of paired-pulse low-frequency stimulation. Selective potentiation of mGluR5 had no effect on LTP induced by suprathreshold TBS or saturated LTP. The finding that potentiation of mGluR5-mediated responses to stimulation of glutamatergic afferents enhances both LTP and LTD and supports the hypothesis that the activation of mGluR5 by endogenous glutamate contributes to both forms of plasticity. Furthermore, two systemically active mGluR5 PAMs enhanced performance in the Morris water maze, a measure of hippocampus-dependent spatial learning. Discovery of small molecules that enhance both LTP and LTD in an activity-appropriate manner shows a unique action on synaptic plasticity that may provide a novel approach for the treatment of impaired cognitive function.

  10. Novel p21-Activated Kinase 4 (PAK4) Allosteric Modulators Overcome Drug Resistance and Stemness in Pancreatic Ductal Adenocarcinoma.

    PubMed

    Aboukameel, Amro; Muqbil, Irfana; Senapedis, William; Baloglu, Erkan; Landesman, Yosef; Shacham, Sharon; Kauffman, Michael; Philip, Philip A; Mohammad, Ramzi M; Azmi, Asfar S

    2017-01-01

    The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases and is found to be overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells but not in normal human pancreatic ductal epithelia (HPDE). Gene copy number amplification studies in PDAC patient cohorts confirmed PAK4 amplification making it an attractive therapeutic target in PDAC. We investigated the antitumor activity of novel PAK4 allosteric modulators (PAM) on a panel of PDAC cell lines and chemotherapy-resistant flow-sorted PDAC cancer stem cells (CSC). The toxicity and efficacy of PAMs were evaluated in multiple subcutaneous mouse models of PDAC. PAMs (KPT-7523, KPT-7189, KPT-8752, KPT-9307, and KPT-9274) show antiproliferative activity in vitro against different PDAC cell lines while sparing normal HPDE. Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in PDAC. PAMs inhibited proliferation and antiapoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes containing vimentin. PAMs disrupted CSC spheroid formation through suppression of PAK4. Moreover, PAMs synergize with gemcitabine and oxaliplatin in vitro KPT-9274, currently in a phase I clinical trial (clinicaltrials.gov; NCT02702492), possesses desirable pharmacokinetic properties and is well tolerated in mice with the absence of any signs of toxicity when 200 mg/kg daily is administered either intravenously or orally. KPT-9274 as a single agent showed remarkable antitumor activity in subcutaneous xenograft models of PDAC cell lines and CSCs. These proof-of-concept studies demonstrated the antiproliferative effects of novel PAMs in PDAC and warrant further clinical investigations. Mol Cancer Ther; 16(1); 76-87. ©2016 AACR.

  11. Discovery of VU0409551/JNJ-46778212: An mGlu5 Positive Allosteric Modulator Clinical Candidate Targeting Schizophrenia

    PubMed Central

    2015-01-01

    Herein, we report the structure–activity relationship of a novel series of (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanones as potent, selective, and orally bioavailable metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). On the basis of its robust in vitro potency and in vivo efficacy in multiple preclinical models of multiple domains of schizophrenia, coupled with a good DMPK profile and an acceptable therapeutic window, 17a (VU0409551/JNJ-46778212) was selected as a candidate for further development. PMID:26157544

  12. Identification of selective agonists and positive allosteric modulators for µ- and δ-opioid receptors from a single high-throughput screen.

    PubMed

    Burford, Neil T; Wehrman, Tom; Bassoni, Daniel; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Alt, Andrew

    2014-10-01

    Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.

  13. Potentiating mGluR5 function with a positive allosteric modulator enhances adaptive learning.

    PubMed

    Xu, Jian; Zhu, Yongling; Kraniotis, Stephen; He, Qionger; Marshall, John J; Nomura, Toshihiro; Stauffer, Shaun R; Lindsley, Craig W; Conn, P Jeffrey; Contractor, Anis

    2013-07-18

    Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity and has been associated with several neuropathological disorders. Previous work has shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction and spatial reversal learning, suggesting that mGluR5 signaling is required for different forms of adaptive learning. Here, we tested whether ADX47273, a selective positive allosteric modulator (PAM) of mGluR5, can enhance adaptive learning in mice. We found that systemic administration of the ADX47273 enhanced reversal learning in the Morris Water Maze, an adaptive task. In addition, we found that ADX47273 had no effect on single-session and multi-session extinction, but administration of ADX47273 after a single retrieval trial enhanced subsequent fear extinction learning. Together these results demonstrate a role for mGluR5 signaling in adaptive learning, and suggest that mGluR5 PAMs represent a viable strategy for treatment of maladaptive learning and for improving behavioral flexibility.

  14. FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor.

    PubMed

    Lecat-Guillet, Nathalie; Monnier, Carine; Rovira, Xavier; Kniazeff, Julie; Lamarque, Laurent; Zwier, Jurriaan M; Trinquet, Eric; Pin, Jean-Philippe; Rondard, Philippe

    2017-03-06

    The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

  15. 3-(Imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers: a novel series of mGluR2 positive allosteric modulators.

    PubMed

    Zhang, Lei; Rogers, Bruce N; Duplantier, Allen J; McHardy, Stanley F; Efremov, Ivan; Berke, Helen; Qian, Weimin; Zhang, Andy Q; Maklad, Noha; Candler, John; Doran, Angela C; Lazzaro, John T; Ganong, Alan H

    2008-10-15

    The synthesis and structure-activity relationship (SAR) of a novel series of 3-(imidazolyl methyl)-3-aza-bicyclo[3.1.0]hexan-6-yl)methyl ethers, derived from a high throughput screening (HTS), are described. Subsequent optimization led to identification of potent, metabolically stable and orally available mGluR2 positive allosteric modulators (PAMs).

  16. Discovery and Preclinical Evaluation of BMS-955829, a Potent Positive Allosteric Modulator of mGluR5

    PubMed Central

    2016-01-01

    Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5) are of interest due to their potential therapeutic utility in schizophrenia and other cognitive disorders. Herein we describe the discovery and optimization of a novel oxazolidinone-based chemotype to identify BMS-955829 (4), a compound with high functional PAM potency, excellent mGluR5 binding affinity, low glutamate fold shift, and high selectivity for the mGluR5 subtype. The low fold shift and absence of agonist activity proved critical in the identification of a molecule with an acceptable preclinical safety profile. Despite its low fold shift, 4 retained efficacy in set shifting and novel object recognition models in rodents. PMID:26985317

  17. Dihydrothiazolopyridone Derivatives as a Novel Family of Positive Allosteric Modulators of the Metabotropic Glutamate 5 (mGlu5) Receptor

    PubMed Central

    Bartolomé-Nebreda, José Manuel; Conde-Ceide, Susana; Delgado, Francisca; Iturrino, Laura; Pastor, Joaquín; Pena, Miguel Ángel; Trabanco, Andrés A.; Tresadern, Gary; Wassvik, Carola M.; Stauffer, Shaun R.; Jadhav, Satyawan; Gogi, Kiran; Vinson, Paige N.; Noetzel, Meredith J.; Days, Emily; Weaver, C. David; Lindsley, Craig W.; Niswender, Colleen M.; Jones, Carrie K.; Conn, P. Jeffrey; Rombouts, Frederik; Lavreysen, Hilde; Macdonald, Gregor J.; Mackie, Claire; Steckler, Thomas

    2014-01-01

    Starting from a singleton chromanone high throughput screening (HTS) hit, we describe a focused medicinal chemistry optimization effort leading to the identification of a novel series of phenoxymethyl-dihydrothiazolopyridone derivatives as selective positive allosteric modulators (PAMs) of the metabotropic glutamate 5 (mGlu5) receptor. These dihydrothiazolopyridones potentiate receptor responses in recombinant systems. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) evaluation allowed us to select compound 16a for its assessment in a preclinical animal screen of possible antipsychotic activity. 16a was able to reverse amphetamine-induced hyperlocomotion in rats in a dose-dependent manner without showing any significant motor impairment or overt neurological side effects at comparable doses. Evolution of our medicinal chemistry program, structure activity, and properties relationships (SAR and SPR) analysis as well as a detailed profile for optimized mGlu5 receptor PAM 16a are described. PMID:23947773

  18. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

    PubMed

    Williams, Dustin K; Wang, Jingyi; Papke, Roger L

    2011-10-15

    Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.

  19. CGP7930: a positive allosteric modulator of the GABAB receptor.

    PubMed

    Adams, C L; Lawrence, A J

    2007-01-01

    CGP7930 (3-(3',5'-Di-tert-butyl-4'-hydroxy)phenyl-2,2-dimethylpropanol) is a positive allosteric modulator of the metabotropic GABAB receptor. CGP7930 has been found to modulate the GABAB receptor in the open, or high affinity, state increasing agonist affinity for the receptor and signal transduction efficacy following agonist stimulation. The GABAB heteromeric subunit B2, involved in signal transduction but not ligand binding, seems to be the site of action of CGP7930 and similar allosteric modulators. When administered alone in naïve animals, CGP7930 acts as an anxiolytic in rodents without other overt behavioral effects and has also been demonstrated to reduce self-administration of nicotine, cocaine, or alcohol in rodents, suggesting that "fine tuning" of the GABAB receptor by positive allosteric modulators may be able to regulate abuse of these drugs. Baclofen, the GABAB agonist, is currently finding use in treating addiction and various other disorders, but this can result in off-target effects and tolerance. CGP7930 when co-administered with baclofen enhances its potency, which could in theory minimize deleterious effects. Further study of CGP7930 is required, but this compound, and others like it, holds potential in a clinical setting.

  20. [GABAB receptor as therapeutic target for drug addiction: from baclofen to positive allosteric modulators].

    PubMed

    Agabio, Roberta; Colombo, Giancarlo

    2015-01-01

    The present paper summarizes experimental and clinical data indicating the therapeutic potential of the GABAB receptor agonist, baclofen, in the treatment of alcohol use disorder (AUD) and substance use disorder (SUD). Multiple preclinical studies have demonstrated the ability of baclofen to suppress alcohol drinking (including binge- and relapse-like drinking), oral alcohol self-administration, and intravenous self-administration of cocaine, nicotine, amphetamine, methamphetamine, morphine, and heroin in rodents. Some randomized, controlled trials (RCTs) and case reports support the efficacy of baclofen in suppressing alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. Data from RCTs and open studies investigating baclofen efficacy on SUD are currently less conclusive. Interest in testing high doses of baclofen in AUD and SUD treatment has recently emerged. Preclinical research has extended the anti-addictive properties of baclofen to positive allosteric modulators of the GABAB receptor (GABAB PAMs). In light of their more favourable side effect profile (compared to baclofen), GABAB PAMs may represent a major step forward in a GABAB receptor-based pharmacotherapy of AUD and SUD.

  1. M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss

    PubMed Central

    Bradley, Sophie J.; Bourgognon, Julie-Myrtille; Sanger, Helen E.; Verity, Nicholas; Mogg, Adrian J.; White, David J.; Butcher, Adrian J.; Moreno, Julie A.; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Wess, Jurgen; Pawlak, Robert; Read, David J.; Sexton, Patrick M.; Broad, Lisa M.; Steinert, Joern R.; Mallucci, Giovanna R.; Felder, Christian C.

    2016-01-01

    The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD. PMID:27991860

  2. M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

    PubMed

    Bradley, Sophie J; Bourgognon, Julie-Myrtille; Sanger, Helen E; Verity, Nicholas; Mogg, Adrian J; White, David J; Butcher, Adrian J; Moreno, Julie A; Molloy, Colin; Macedo-Hatch, Timothy; Edwards, Jennifer M; Wess, Jurgen; Pawlak, Robert; Read, David J; Sexton, Patrick M; Broad, Lisa M; Steinert, Joern R; Mallucci, Giovanna R; Christopoulos, Arthur; Felder, Christian C; Tobin, Andrew B

    2017-02-01

    The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

  3. Novel bivalent positive allosteric modulators of AMPA receptor.

    PubMed

    Lavrov, M I; Grigor'ev, V V; Bachurin, S O; Palyulin, V A; Zefirov, N S

    2015-01-01

    A positive allosteric modulator of AMPA receptors has been designed using computer-aided molecular modeling techniques. It possessed a record high experimentally confirmed potency in the picomolar concentration range and belongs to a new type of bivalent AMPA receptor ligands containing bicyclo[3.3.1]nonane scaffold. The suggested structure could serve as a basis for further optimization and development of drugs for the treatment of neurodegenerative diseases, cognition enhancement, and improvement of memory.

  4. Discovery of a Negative Allosteric Modulator of GABAB Receptors

    PubMed Central

    2014-01-01

    Initialized from the scaffold of CGP7930, an allosteric agonist of GABAB receptors, a series of noncompetitive antagonists were discovered. Among these compounds, compounds 3, 6, and 14 decreased agonist GABA-induced maximal effect of IP3 production in HEK293 cells overexpressing GABAB receptors and Gqi9 proteins without changing the EC50. Compounds 3, 6, and 14 not only inhibited agonist baclofen-induced ERK1/2 phosphorylation but also blocked CGP7930-induced ERK1/2 phosphorylation in HEK293 cells overexpressing GABAB receptors. The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABAB receptors. The representative compound 14 decreased GABA-induced IP3 production with IC50 of 37.9 μM and had no effect on other GPCR Class C members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding sites of GABAB receptors, demonstrating that compound 14 negatively modulated GABAB receptors activity as a negative allosteric modulator. PMID:25050158

  5. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-07-02

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.

  6. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    PubMed Central

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  7. Sex-Dependent Anti-Stress Effect of an α5 Subunit Containing GABAA Receptor Positive Allosteric Modulator

    PubMed Central

    Piantadosi, Sean C.; French, Beverly J.; Poe, Michael M.; Timić, Tamara; Marković, Bojan D.; Pabba, Mohan; Seney, Marianne L.; Oh, Hyunjung; Orser, Beverley A.; Savić, Miroslav M.; Cook, James M.; Sibille, Etienne

    2016-01-01

    Rationale: Current first-line treatments for stress-related disorders such as major depressive disorder (MDD) act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2’F-R-CH3 (denoted “α5-PAM”), a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites, has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS) and treated with α5-PAM acutely (30 min prior to assessing behavior) or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as “behavioral emotionality”) across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in the hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusion: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities

  8. Identification of glutathione conjugates of acetylene-containing positive allosteric modulators of metabotropic glutamate receptor subtype 5.

    PubMed

    Zhuo, Xiaoliang; Huang, Xiaohua Stella; Degnan, Andrew P; Snyder, Lawrence B; Yang, Fukang; Huang, Hong; Shu, Yue-Zhong; Johnson, Benjamin M

    2015-04-01

    A recent medicinal chemistry campaign to identify positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) led to the discovery of potent compounds featuring an oxazolidinone structural core flanked by biaryl acetylene and haloaryl moieties. However, biotransformation studies of some of these mGluR5 PAMs demonstrated the formation of glutathione (GSH) conjugates. The conjugates in question were formed independently of NADPH as the main products in liver microsomes and liver cytosol (rat and human) and exhibited masses that were 307 u greater than their respective substrates, indicating the involvement of a reductive step in the formation of these metabolites. To further characterize the relevant metabolic sequences, GSH conjugates of (4R,5R)-5-(3-fluorophenyl)-4-(5-(pyrazin-2-ylethynyl)pyridin-3-yl)oxazolidin-2-one and (4R,5R)-5-(4-fluorophenyl)-4-(6-((3-fluoropyridin-2-yl)ethynyl)pyridin-2-yl)oxazolidin-2-one were biosynthesized and isolated. Subsequent analysis by NMR showed that GSH had reacted with the acetylene carbon atoms of these mGluR5 PAMs, suggesting a conjugate addition mechanism and implicating cytosolic and microsomal GSH S-transferases (GSTs) in catalysis. Interestingly, five closely related mGluR5 PAMs were not similarly prone to the formation of GSH conjugates in vitro. These compounds also featured acetylenes, but were flanked by either phenyl or cyclohexyl rings, which indicated that the formation of GSH conjugates was influenced by proximal functional groups that modulated the electron density of the triple bond and/or differences in enzyme-substrate specificity. These results informed an ongoing drug-discovery effort to identify mGluR5 PAMs with drug-like properties and a low risk of reactivity with endogenous thiols.

  9. Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors.

    PubMed

    Thomsen, Morten S; Mikkelsen, Jens D

    2012-10-01

    Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL-3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine-induced receptor up-regulation, suggesting that nicotine and A-582941 induce up-regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU-120596 inhibits up-regulation of the α7 nAChR induced by 10 mg/kg A-582941, as measured by [(125)I]-bungarotoxin autoradiography, whereas 1 mg/kg AVL-3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A-582941-induced up-regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist-dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.

  10. Low-power DAC-less PAM-4 transmitter using a cascaded microring modulator.

    PubMed

    Dubé-Demers, Raphaël; LaRochelle, Sophie; Shi, Wei

    2016-11-15

    Future super-computer interconnect systems and data centers request ultrahigh data rate links at low cost and power consumption, for which transmitters with a high level of integration and spectral efficient formats are key components. We report 60 Gb/s pulse-amplitude modulation (PAM-4) of an optical signal using a dual-microring silicon photonics circuit, making a low-power, digital-to-analog converter (DAC)-less PAM modulator. The power consumption is evaluated below 100 fJ/bit, including thermal adjustments. To the best of our knowledge, these results feature the lowest reported power consumption for PAM signaling in a DAC-less scheme for data rate beyond 40 Gb/s.

  11. Tetrahydronaphthyridine and Dihydronaphthyridinone Ethers As Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 5 (mGlu5)

    PubMed Central

    2015-01-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Starting from an acetylene-based lead from high throughput screening, an evolved bicyclic dihydronaphthyridinone was identified. We describe further refinements leading to both dihydronaphthyridinone and tetrahydronaphthyridine mGlu5 PAMs containing an alkoxy-based linkage as an acetylene replacement. Exploration of several structural features including western pyridine ring isomers, positional amides, linker connectivity/position, and combinations thereof, reveal that these bicyclic modulators generally exhibit steep SAR and within specific subseries display a propensity for pharmacological mode switching at mGlu5 as well as antagonist activity at mGlu3. Structure–activity relationships within a dihydronaphthyridinone subseries uncovered 12c (VU0405372), a selective mGlu5 PAM with good in vitro potency, low glutamate fold-shift, acceptable DMPK properties, and in vivo efficacy in an amphetamine-based model of psychosis. PMID:24914612

  12. Development of 1H-Pyrazolo[3,4-b]pyridines as Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators.

    PubMed

    Hill, Matthew D; Fang, Haiquan; Brown, Jeffrey M; Molski, Thaddeus; Easton, Amy; Han, Xiaojun; Miller, Regina; Hill-Drzewi, Melissa; Gallagher, Lizbeth; Matchett, Michele; Gulianello, Michael; Balakrishnan, Anand; Bertekap, Robert L; Santone, Kenneth S; Whiterock, Valerie J; Zhuo, Xiaoliang; Bronson, Joanne J; Macor, John E; Degnan, Andrew P

    2016-12-08

    The metabotropic glutamate receptor 5 (mGluR5) is an attractive target for the treatment of schizophrenia due to its role in regulating glutamatergic signaling in association with the N-methyl-d-aspartate receptor (NMDAR). We describe the synthesis of 1H-pyrazolo[3,4-b]pyridines and their utility as mGluR5 positive allosteric modulators (PAMs) without inherent agonist activity. A facile and convergent synthetic route provided access to a structurally diverse set of analogues that contain neither the aryl-acetylene-aryl nor aryl-methyleneoxy-aryl elements, the predominant structural motifs described in the literature. Binding studies suggest that members of our new chemotype do not engage the receptor at the MPEP and CPPHA mGluR5 allosteric sites. SAR studies culminated in the first non-MPEP site PAM, 1H-pyrazolo[3,4-b]pyridine 31 (BMT-145027), to improve cognition in a preclinical rodent model of learning and memory.

  13. Homology modeling, docking, and molecular dynamics simulation of the receptor GALR2 and its interactions with galanin and a positive allosteric modulator.

    PubMed

    Hui, Wen-Qi; Cheng, Qi; Liu, Tian-Yu; Ouyang, Qin

    2016-04-01

    Galanin receptor type 2 (GALR2) is a class A G-protein-coupled receptor (GPCR), and it has been reported that orthosteric ligands and positive allosteric modulators (PAMs) of GALR2 could potentially be used to treat epilepsy. So far, the X-ray structure of this receptor has not been resolved, and knowledge of the 3D structure of GALR2 may prove informative in attempts to design novel ligands and to explore the mechanism for the allosteric modulation of this receptor. In this study, homology modeling was used to obtain several GALR2 models using known templates. ProSA-web Z-scores and Ramachandran plots as well as pre-screening against a test dataset of known compounds were all utilized to select the best model of GALR2. Molecular dockings of galanin (a peptide) and a nonpeptide ligand were carried out to choose the (GALR2 model)-galanin complex that showed the closest agreement with the corresponding experimental data. Finally, a 50-ns MD simulation was performed to study the interactions between the GALR2 model and the synthetic and endogenous ligands. The results from docking and MD simulation showed that, besides the reported residues, Tyr160(4.60), Ile105(3.32), Ala274(7.35), and Tyr163(ECL2) also appear to play important roles in the binding of galanin. The potential allosteric binding pockets in the GALR2 model were then investigated via MD simulation. The results indicated that the mechanism for the allosteric modulation caused by PAMs is the binding of the PAM at pocket III, which is formed by galanin, ECL2, TM2, TM3, and ECL1; this results in the disruption of the Na(+)-binding site and/or the Na(+) ion pathway, leading to GALR2 agonism.

  14. Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor

    PubMed Central

    Kahsai, Alem W.; Wisler, James W.; Lee, Jungmin; Ahn, Seungkirl; Cahill, Thomas J.; Dennison, S. Moses; Staus, Dean P.; Thomsen, Alex R. B.; Anasti, Kara M.; Pani, Biswaranjan; Wingler, Laura M.; Desai, Hemant; Bompiani, Kristin M.; Strachan, Ryan T.; Qin, Xiaoxia; Alam, S. Munir; Sullenger, Bruce A.; Lefkowitz, Robert J.

    2016-01-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs. PMID:27398998

  15. Biased mGlu5 positive allosteric modulators provide in vivo efficacy without potentiating mGlu5 modulation of NMDAR currents

    PubMed Central

    Rook, Jerri M.; Xiang, Zixiu; Lv, Xiaohui; Ghoshal, Ayan; Dickerson, Jonathan W.; Bridges, Thomas M.; Johnson, Kari A.; Foster, Daniel J.; Gregory, Karen J.; Vinson, Paige N.; Thompson, Analisa D.; Byun, Nellie; Collier, Rebekah L.; Bubser, Michael; Nedelcovych, Michael T.; Gould, Robert W.; Stauffer, Shaun R.; Daniels, J. Scott; Niswender, Colleen M.; Lavreysen, Hilde; Mackie, Claire; Conde-Ceide, Susana; Alcazar, Jesus; Bartolomé-Nebreda, José M.; Macdonald, Gregor J.; Steckler, Thomas; Jones, Carrie K.; Lindsley, Craig W.; Conn, P. Jeffrey

    2015-01-01

    Summary Schizophrenia is associated with disruptions in N-methyl-D-aspartate glutamate receptor subtype (NMDAR)-mediated excitatory synaptic signaling. The metabotropic glutamate receptor subtype 5 (mGlu5) is a closely associated signaling partner with NMDARs and regulates NMDAR function in forebrain regions implicated in the pathology of schizophrenia. Efficacy of mGlu5 positive allosteric modulators (PAMs) in animal models of psychosis and cognition was previously attributed to potentiation of NMDAR function. To directly test this hypothesis, we identified VU0409551 as a novel mGlu5 PAM that exhibits distinct stimulus bias and selectively potentiates mGlu5 coupling to Gαq–mediated signaling but not mGlu5 modulation of NMDAR currents or NMDAR-dependent synaptic plasticity in the rat hippocampus. Interestingly, VU0409551 produced robust antipsychotic-like and cognition-enhancing activity in animal models. These data provide surprising new mechanistic insights into the actions of mGlu5 PAMs and suggest that modulation of NMDAR currents is not critical for in vivo efficacy. PMID:25937172

  16. Novel PAMs Targeting NMDAR GluN2A Subunit.

    PubMed

    Xiang, Zixiu; Conn, P Jeffrey

    2016-03-02

    In this issue of Neuron, Hackos et al. (2016) report the discovery of novel positive allosteric modulators that are highly selective for GluN2A-containing NMDA receptors. This novel class of PAMs shows distinct effects on synaptic plasticity.

  17. Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

    PubMed

    Malysz, John; Grønlien, Jens H; Timmermann, Daniel B; Håkerud, Monika; Thorin-Hagene, Kirsten; Ween, Hilde; Trumbull, Jonathan D; Xiong, Yongli; Briggs, Clark A; Ahring, Philip K; Dyhring, Tino; Gopalakrishnan, Murali

    2009-08-01

    Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant

  18. Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function.

    PubMed

    Hackos, David H; Lupardus, Patrick J; Grand, Teddy; Chen, Yelin; Wang, Tzu-Ming; Reynen, Paul; Gustafson, Amy; Wallweber, Heidi J A; Volgraf, Matthew; Sellers, Benjamin D; Schwarz, Jacob B; Paoletti, Pierre; Sheng, Morgan; Zhou, Qiang; Hanson, Jesse E

    2016-03-02

    To enhance physiological function of NMDA receptors (NMDARs), we identified positive allosteric modulators (PAMs) of NMDARs with selectivity for GluN2A subunit-containing receptors. X-ray crystallography revealed a binding site at the GluN1-GluN2A dimer interface of the extracellular ligand-binding domains (LBDs). Despite the similarity between the LBDs of NMDARs and AMPA receptors (AMPARs), GluN2A PAMs with good selectivity against AMPARs were identified. Potentiation was observed with recombinant triheteromeric GluN1/GluN2A/GluN2B NMDARs and with synaptically activated NMDARs in brain slices from wild-type (WT), but not GluN2A knockout (KO), mice. Individual GluN2A PAMs exhibited variable degrees of glutamate (Glu) dependence, impact on NMDAR Glu EC50, and slowing of channel deactivation. These distinct PAMs also exhibited differential impacts during synaptic plasticity induction. The identification of a new NMDAR modulatory site and characterization of GluN2A-selective PAMs provide powerful molecular tools to dissect NMDAR function and demonstrate the feasibility of a therapeutically desirable type of NMDAR enhancement.

  19. High gain preamplifier module (PAM) engineering prototype for the National Ignition Facility (NIF) laser system

    SciTech Connect

    Braucht, J; Browning, D; Crane, J K; Crawford, J; Deadrick, F J; Hawkins, S; Jones, B; Martinez, M; Penko, F; Skulina, M; Tilley, R; Wing, R

    1998-10-29

    We describe recent results and developments in the preamplifier module (PAM) engineering prototype located in NIF's front end or Optical Pulse Generation (OPG) system. This prototype uses the general laser design developed on a physics testbed and integrates NIP type packaging as well as controls and diagnostics. We will present laser, mechanical and electrical hardware designed and built to date as well as laser energetics measurements.

  20. A Cannabinoid CB1 Receptor-Positive Allosteric Modulator Reduces Neuropathic Pain in the Mouse with No Psychoactive Effects

    PubMed Central

    Ignatowska-Jankowska, Bogna M; Baillie, Gemma L; Kinsey, Steven; Crowe, Molly; Ghosh, Sudeshna; Owens, Robert A; Damaj, Imad M; Poklis, Justin; Wiley, Jenny L; Zanda, Matteo; Zanato, Chiara; Greig, Iain R; Lichtman, Aron H; Ross, Ruth A

    2015-01-01

    The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ9-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [3H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [35S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects. PMID:26052038

  1. Discovery and SAR of a novel series of non-MPEP site mGlu₅ PAMs based on an aryl glycine sulfonamide scaffold.

    PubMed

    Rodriguez, Alice L; Zhou, Ya; Williams, Richard; Weaver, C David; Vinson, Paige N; Dawson, Eric S; Steckler, Thomas; Lavreysen, Hilde; Mackie, Claire; Bartolomé, José M; Macdonald, Gregor J; Daniels, J Scott; Niswender, Colleen M; Jones, Carrie K; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

    2012-12-15

    Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu(5)) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu(5) PAM chemotype.

  2. Allosteric Modulation: An Alternate Approach Targeting the Cannabinoid CB1 Receptor.

    PubMed

    Nguyen, Thuy; Li, Jun-Xu; Thomas, Brian F; Wiley, Jenny L; Kenakin, Terry P; Zhang, Yanan

    2016-11-23

    The cannabinoid CB1 receptor is a G protein coupled receptor and plays an important role in many biological processes and physiological functions. A variety of CB1 receptor agonists and antagonists, including endocannabinoids, phytocannabinoids, and synthetic cannabinoids, have been discovered or developed over the past 20 years. In 2005, it was discovered that the CB1 receptor contains allosteric site(s) that can be recognized by small molecules or allosteric modulators. A number of CB1 receptor allosteric modulators, both positive and negative, have since been reported and importantly, they display pharmacological characteristics that are distinct from those of orthosteric agonists and antagonists. Given the psychoactive effects commonly associated with CB1 receptor agonists and antagonists/inverse agonists, allosteric modulation may offer an alternate approach to attain potential therapeutic benefits while avoiding inherent side effects of orthosteric ligands. This review details the complex pharmacological profiles of these allosteric modulators, their structure-activity relationships, and efforts in elucidating binding modes and mechanisms of actions of reported CB1 allosteric modulators. The ultimate development of CB1 receptor allosteric ligands could potentially lead to improved therapies for CB1-mediated neurological disorders.

  3. Allosteric Modulation of Metabotropic Glutamate Receptors: Structural Insights and Therapeutic Potential

    PubMed Central

    Gregory, Karen J.; Dong, Elizabeth N.; Meiler, Jens; Conn, P. Jeffrey

    2010-01-01

    Allosteric modulation of G protein-coupled receptors (GPCRs) represents a novel approach to the development of probes and therapeutics that is expected to enable subtype-specific regulation of central nervous system target receptors. The metabotropic glutamate receptors (mGlus) are class C GPCRs that play important neuromodulatory roles throughout the brain, as such they are attractive targets for therapeutic intervention for a number of psychiatric and neurological disorders including anxiety, depression, Fragile X Syndrome, Parkinson’s disease and schizophrenia. Over the last fifteen years, selective allosteric modulators have been identified for many members of the mGlu family. The vast majority of these allosteric modulators are thought to bind within the transmembrane-spanning domains of the receptors to enhance or inhibit functional responses. A combination of mutagenesis-based studies and pharmacological approaches are beginning to provide a better understanding of mGlu allosteric sites. Collectively, when mapped onto a homology model of the different mGlu subtypes based on the β2-adrenergic receptor, the previous mutagenesis studies suggest commonalities in the location of allosteric sites across different members of the mGlu family. In addition, there is evidence for multiple allosteric binding pockets within the transmembrane region that can interact to modulate one another. In the absence of a class C GPCR crystal structure, this approach has shown promise with respect to the interpretation of mutagenesis data and understanding structure-activity relationships of allosteric modulator pharmacophores. PMID:20637216

  4. 3-Furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic receptor, reverses schizophrenia-like cognitive and social deficits in rats.

    PubMed

    Potasiewicz, Agnieszka; Hołuj, Małgorzata; Kos, Tomasz; Popik, Piotr; Arias, Hugo R; Nikiforuk, Agnieszka

    2017-02-01

    The cognitive impairments and negative symptoms experienced by schizophrenia patients still await effective treatment. Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) have gain considerable attention in this regard. It has been recently proposed that positive allosteric modulators (PAMs) of α7 nAChRs may represent an alternative strategy to that based on orthosteric agonists. The aim of the present study is to evaluate the efficacy of PAM-2 (3-furan-2-yl-N-p-tolyl-acrylamide) against cognitive deficits and negative-like symptoms in a rat model of schizophrenia based on administration of ketamine, a NMDAR antagonist. The activity of PAM-2 was compared to that elicited by DMXBA, an α7 nAChR partial agonist. For this purpose, the attentional set-shifting task (ASST) and the novel object recognition task (NORT) were used. The efficacies of PAM-2 and DMXBA against ketamine-induced social withdrawal were assessed using the social interaction test (SIT). The results demonstrated that PAM-2 and DMXBA ameliorated ketamine-induced cognitive impairments on the ASST and NORT as well as produced pro-social activities in the SIT. Moreover, the co-administration of inactive doses of PAM-2 and antipsychotic drugs, clozapine or risperidone, reversed ketamine-induced deficits. The present findings provide further support for the concept that α7-PAMs could be used either alone or in combination with antipsychotics for schizophrenia therapy.

  5. In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

    PubMed

    Zysk, John R; Spear, Nathan; Fieles, William; Stein, Mark M; Sygowski, Linda S; King, Megan M; Hoesch, Valerie; Hastings, Richard; Brockel, Becky; Do, Mylinh; Ström, Peter; Gadient, Reto; Chhajlani, Vijay; Elmore, Charles S; Maier, Donna L

    2013-03-01

    The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important

  6. New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide.

    PubMed

    Nakane, Atsushi; Gotoh, Yusuke; Ichihara, Junji; Nagata, Hidetaka

    2015-12-15

    The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide.

  7. N-Aryl Piperazine Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators Possess Efficacy in Preclinical Models of NMDA Hypofunction and Cognitive Enhancement

    PubMed Central

    Gregory, K.J.; Herman, E.J.; Ramsey, A.J.; Hammond, A.S.; Byun, N.E.; Stauffer, S.R.; Manka, J.T.; Jadhav, S.; Bridges, T.M.; Weaver, C.D.; Niswender, C.M.; Steckler, T.; Drinkenburg, W.H.; Ahnaou, A.; Lavreysen, H.; Macdonald, G.J.; Bartolomé, J.M.; Mackie, C.; Hrupka, B.J.; Caron, M.G.; Daigle, T.L.; Lindsley, C.W.; Conn, P.J.

    2013-01-01

    Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca2+ mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel

  8. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  9. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  10. Allosteric modulators of the hERG K{sup +} channel

    SciTech Connect

    Yu, Zhiyi Klaasse, Elisabeth Heitman, Laura H. IJzerman, Adriaan P.

    2014-01-01

    Drugs that block the cardiac K{sup +} channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K{sup +} channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [{sup 3}H]astemizole and [{sup 3}H]dofetilide to the hERG K{sup +} channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC{sub 50} values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC{sub 50} values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K{sup +} channel, which is discussed in the light of findings on other ion channels. - Highlights: • Allosteric modulators on the hERG K{sup +} channel were evaluated in binding assays. • LUF6200 was identified as a potent allosteric inhibitor. • Potassium ions were found to behave as allosteric enhancers. • Positive cooperativity and distinct allosteric sites for them were proposed.

  11. Ignavine: a novel allosteric modulator of the μ opioid receptor

    PubMed Central

    Ohbuchi, Katsuya; Miyagi, Chika; Suzuki, Yasuyuki; Mizuhara, Yasuharu; Mizuno, Keita; Omiya, Yuji; Yamamoto, Masahiro; Warabi, Eiji; Sudo, Yuka; Yokoyama, Akinobu; Miyano, Kanako; Hirokawa, Takatsugu; Uezono, Yasuhito

    2016-01-01

    Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy. PMID:27530869

  12. Optical coherence photoacoustic microscopy (OC-PAM) with an intensity-modulated continuous-wave broadband light source

    NASA Astrophysics Data System (ADS)

    Liu, Xiaojing; Wen, Rong; Li, Yiwen; Jiao, Shuliang

    2016-06-01

    We developed an optical coherence photoacoustic microscopy system using an intensity-modulated continuous-wave superluminescent diode with a center wavelength of 840 nm. The system can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously. Compared to the system with a pulsed light source, this system is able to achieve OCT imaging with quality as high as conventional spectral-domain OCT. Since both of the OCT and PAM images are generated from the same group of photons, they are intrinsically registered in the lateral directions. The system was tested for multimodal imaging the vasculature of mouse ear in vivo by using gold nanorods as contrast agent for PAM, as well as excised porcine eyes ex vivo. The OCT and PAM images showed complimentary information of the sample.

  13. Experimental investigation of PAM, CAP and DMT modulations efficiency over a double-step-index polymer optical fiber

    NASA Astrophysics Data System (ADS)

    Stepniak, G.; Siuzdak, J.

    2014-08-01

    Investigated was the transmission over step index POF that is 50 m/100 m long with a red DVD laser as a source and a Si p-i-n commercial photo-detector. The maximum bit rates of three modulation formats: PAM, CAP and DMT were sought. Their constellation sizes and symbol rates were varied in order to maximize FEC limited throughputs for each modulation whereas the laser operating point and relevant optical powers were maintained constant for all the modulations. The maximum throughputs were similar for PAM and CAP, namely 3.3/2 Gbit/s for PAM, and 3.15/2.1 for CAP for 50/100 m fiber, respectively. The bit rates for DMT were 2.65/1.65 Gbit/s for the respective lengths of the fiber. We attribute the inferior performance of DMT to its high value of peak to average power ratio.

  14. Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry.

    PubMed

    Herlory, Olivier; Bonzom, Jean-Marc; Gilbin, Rodolphe

    2013-09-15

    Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency ( [Formula: see text] , EC50=303 ± 64 μg UL(-1) after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142 ± 98 μg UL(-1) after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae.

  15. Resolving the contribution of the uncoupled phycobilisomes to cyanobacterial pulse-amplitude modulated (PAM) fluorometry signals.

    PubMed

    Acuña, Alonso M; Snellenburg, Joris J; Gwizdala, Michal; Kirilovsky, Diana; van Grondelle, Rienk; van Stokkum, Ivo H M

    2016-01-01

    Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.

  16. Antipsychotic drug-like effects of the selective M4 muscarinic acetylcholine receptor positive allosteric modulator VU0152100.

    PubMed

    Byun, Nellie E; Grannan, Michael; Bubser, Michael; Barry, Robert L; Thompson, Analisa; Rosanelli, John; Gowrishankar, Raajaram; Kelm, Nathaniel D; Damon, Stephen; Bridges, Thomas M; Melancon, Bruce J; Tarr, James C; Brogan, John T; Avison, Malcolm J; Deutch, Ariel Y; Wess, Jürgen; Wood, Michael R; Lindsley, Craig W; Gore, John C; Conn, P Jeffrey; Jones, Carrie K

    2014-06-01

    Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis

  17. Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

    PubMed Central

    Byun, Nellie E; Grannan, Michael; Bubser, Michael; Barry, Robert L; Thompson, Analisa; Rosanelli, John; Gowrishankar, Raajaram; Kelm, Nathaniel D; Damon, Stephen; Bridges, Thomas M; Melancon, Bruce J; Tarr, James C; Brogan, John T; Avison, Malcolm J; Deutch, Ariel Y; Wess, Jürgen; Wood, Michael R; Lindsley, Craig W; Gore, John C; Conn, P Jeffrey; Jones, Carrie K

    2014-01-01

    Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis

  18. Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

    PubMed

    Arias, Hugo R; Ravazzini, Federica; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Feuerbach, Dominik; Boffi, Juan C; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puia, Giulia

    2016-07-01

    The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

  19. Distinct effects of mGlu4 receptor positive allosteric modulators at corticostriatal vs. striatopallidal synapses may differentially contribute to their antiparkinsonian action.

    PubMed

    Gubellini, Paolo; Melon, Christophe; Dale, Elena; Doller, Dario; Kerkerian-Le Goff, Lydia

    2014-10-01

    Metabotropic glutamate 4 (mGlu4) receptor is a promising target for the treatment of motor deficits in Parkinson's disease (PD). This is due in part to its localization at key basal ganglia (BG) synapses that become hyperactive in this pathology, particularly striatopallidal synapses. In this context, mGlu4 receptor activation using either orthosteric agonists or positive allosteric modulators (PAMs) improves motor symptoms in rodent PD models in certain conditions. However, literature data show that mGlu4 receptor PAMs have no effect at striatopallidal GABAergic synapses (unless combined with an orthosteric agonist) and on the firing activity of pallidal neurons, and fail to provide significant motor improvement in relevant PD models. This questions the mechanistic hypothesis that mGlu4 receptor PAMs should act at striatopallidal synapses to alleviate PD motor symptoms. To shed light on this issue, we performed brain slice electrophysiology experiments. We show that Lu AF21934, an mGlu4 PAM small-molecule probe-compound, was ineffective at striatopallidal synapses at all concentrations tested, while it significantly inhibited corticostriatal synaptic transmission. Similarly, Lu AF21934 did not affect electrophysiology readouts at striatopallidal synapses in the presence of haloperidol or in 6-hydroxydopamine-lesioned rats. Interestingly, co-application of Lu AF21934 with a glutamate transporter inhibitor revealed a significant inhibitory action at striatopallidal synapses. Possibly, this effect could rely on increased level/permanence of glutamate in the synaptic cleft. Such differential efficacy of mGlu4 receptor PAMs at corticostriatal vs. striatopallidal synapses raises several issues regarding the synaptic target(s) of these drugs in the BG, and challenges the mechanisms by which they alleviate motor deficits in experimental PD models.

  20. High therapeutic potential of positive allosteric modulation of α7 nAChRs in a rat model of traumatic brain injury: Proof-of-concept

    PubMed Central

    Gatson, Joshua W.; Simpkins, James W.; Uteshev, Victor V.

    2015-01-01

    There are currently no clinically-efficacious drug therapies to treat brain damage secondary to traumatic brain injury (TBI). In this proof-of-concept study, we used a controlled cortical impact model of TBI in young adult rats to explore a novel promising approach that utilizes PNU-120596, a previously-reported highly selective Type-II positive allosteric modulator (α7-PAM) of α7 nicotinic acetylcholine receptors (nAChRs). α7-PAMs enhance and prolong α7 nAChR activation, but do not activate α7 nAChRs when administered without an agonist. The rational basis for the use of an α7-PAM as a post-TBI treatment is tripartite and arises from: 1) the intrinsic ability of brain injury to elevate extracellular levels of choline (a ubiquitous cell membrane-building material and a selective endogenous agonist of α7 nAChRs) due to the breakdown of cell membranes near the site and time of injury; 2) the ubiquitous expression of functional α7 nAChRs in neuronal and glial/immune brain cells; and 3) the potent neuroprotective and anti-inflammatory effects of α7 nAChR activation. Therefore, both neuroprotective and anti-inflammatory effects can be achieved post-TBI by targeting only a single player (i.e., the α7 nAChR) using α7-PAMs to enhance the activation of α7 nAChRs by injury-elevated extracellular choline. Our data support this hypothesis and demonstrate that subcutaneous administration of PNU-120596 post-TBI in young adult rats significantly reduces both brain cell damage and reactive gliosis. Therefore, our results introduce post-TBI systemic administration of α7-PAMs as a promising therapeutic intervention that could significantly restrict brain injury post-TBI and facilitate recovery of TBI patients. PMID:25647232

  1. Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

    PubMed Central

    2016-01-01

    Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary (“orthosteric”) site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid–water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand–receptor distance and ligand–receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators. PMID:26841170

  2. FUNCTIONAL INSIGHT INTO DEVELOPMENT OF POSITIVE ALLOSTERIC MODULATORS OF AMPA RECEPTORS

    PubMed Central

    Weeks, Autumn M.; Harms, Jonathan E.; Partin, Kathryn M.; Benveniste, Morris

    2014-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five “subsites” which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites “A” and “B” of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds. PMID:24878241

  3. Functional insight into development of positive allosteric modulators of AMPA receptors.

    PubMed

    Weeks, Autumn M; Harms, Jonathan E; Partin, Kathryn M; Benveniste, Morris

    2014-10-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five "subsites" which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites "A" and "B" of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

  4. Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice

    PubMed Central

    Mitra, Swarup; Mucha, Mckenzie; Khatri, Shailesh N.; Glenon, Richard; Schulte, Marvin K.; Bult-Ito, Abel

    2017-01-01

    Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1–2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD. PMID:28105008

  5. Tuning the endocannabinoid system: allosteric modulators of the CB1 receptor.

    PubMed

    Ross, R A

    2007-11-01

    Cannabinoid CB1 receptor antagonists are novel therapeutics with potential for the treatment of a number of conditions including obesity, nicotine addition and metabolic syndrome. In 2005, Price et al. demonstrated that the cannabinoid CB1 receptor contains an allosteric-binding site which binds synthetic small molecules. In this issue of the British Journal of Pharmacology, Horswill et al. have extended these observations. They demonstrate that a structurally similar small molecule allosterically modulates the cannabinoid CB1 receptor and reduces body weight and food intake in an acute feeding model. Allosteric modulation now contends as a new strategy in the therapeutic exploitation of cannabinoid receptors that may offer certain advantages over the more familiar small molecules targeting the orthosteric site.

  6. Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists.

    PubMed

    Bíró, Tímea; Maksay, Gábor

    2004-06-01

    Allosteric modulation of [3H]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [3H]strychnine and glycine bindings (alpha and beta, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (beta-alanine, taurine and gamma-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 microM propofol. Further, propofol showed positive cooperativity (beta < 1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [3H]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity (beta > 1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.

  7. Neurophysiologic and antipsychotic profiles of TASP0433864, a novel positive allosteric modulator of metabotropic glutamate 2 receptor.

    PubMed

    Hiyoshi, Tetsuaki; Marumo, Toshiyuki; Hikichi, Hirohiko; Tomishima, Yasumitsu; Urabe, Hiroki; Tamita, Tomoko; Iida, Izumi; Yasuhara, Akito; Karasawa, Jun-ichi; Chaki, Shigeyuki

    2014-12-01

    Excess glutamatergic neurotransmission has been implicated in the pathophysiology of schizophrenia, and the activation of metabotropic glutamate 2 (mGlu2) receptor may exert antipsychotic effects by normalizing glutamate transmission. In the present study, we investigated the neurophysiologic and antipsychotic profiles of TASP0433864 [(2S)-2-[(4-tert-butylphenoxy)methyl]-5-methyl-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide], a newly synthesized positive allosteric modulator (PAM) of mGlu2 receptor. TASP0433864 exhibited PAM activity at human and rat mGlu2 receptors with EC50 values of 199 and 206 nM, respectively, without exerting agonist activity at rat mGlu2 receptor. TASP0433864 produced a leftward and upward shift in the concentration-response curve of glutamate-increased guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to mGlu2 receptor. In contrast, TASP0433864 had negligible activities for other mGlu receptors, including mGlu3 receptor, and did not have any affinity for other receptors or transporters. In hippocampal slices, TASP0433864 potentiated an inhibitory effect of DCG-IV [(2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine], a mGlu2/3 receptor agonist, on the field excitatory postsynaptic potentials in the dentate gyrus, indicating that TASP0433864 potentiates the mGlu2 receptor-mediated presynaptic inhibition of glutamate release. Moreover, TASP0433864 inhibited both MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]- and ketamine-increased cortical γ band oscillation in the rat cortical electroencephalogram, which have been considered to reflect the excess activation of cortical pyramidal neurons. The inhibitory effect of TASP0433864 on cortical activation was also observed in the mouse 2-deoxy-glucose uptake study. In a behavioral study, TASP0433864 significantly inhibited both ketamine- and methamphetamine-increased locomotor activities in mice and rats, respectively. Collectively, these

  8. Extracellular Loop 2 of the Free Fatty Acid Receptor 2 Mediates Allosterism of a Phenylacetamide Ago-Allosteric ModulatorS⃞

    PubMed Central

    Smith, Nicola J.; Ward, Richard J.; Stoddart, Leigh A.; Hudson, Brian D.; Kostenis, Evi; Ulven, Trond; Morris, Joanne C.; Tränkle, Christian; Tikhonova, Irina G.; Adams, David R.

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB. PMID:21498659

  9. Modulation of hemoglobin dynamics by an allosteric effector

    PubMed Central

    Maccarini, Marco; Fouquet, Peter; Ho, Nancy T.; Ho, Chien; Makowski, Lee

    2017-01-01

    Abstract Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors. PMID:27977887

  10. CB(1) receptor allosteric modulators display both agonist and signaling pathway specificity.

    PubMed

    Baillie, Gemma L; Horswill, James G; Anavi-Goffer, Sharon; Reggio, Patricia H; Bolognini, Daniele; Abood, Mary E; McAllister, Sean; Strange, Phillip G; Stephens, Gary J; Pertwee, Roger G; Ross, Ruth A

    2013-02-01

    We have previously identified allosteric modulators of the cannabinoid CB(1) receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB(1) receptor agonist [(3)H]CP55940 but producing a decrease in CB(1) receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB(1) receptor activation. We assessed the effect of these compounds on CB(1) receptor agonist-induced [(35)S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB(1) agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [(3)H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding, simulation (Gα(s)-mediated), and inhibition (Gα(i)-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB(1) agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

  11. Differential Modulation of Thresholds for Intracranial Self-Stimulation by mGlu5 Positive and Negative Allosteric Modulators: Implications for Effects on Drug Self-Administration

    PubMed Central

    Cleva, Richard M.; Watterson, Lucas R.; Johnson, Meagan A.; Olive, M. Foster

    2011-01-01

    Pharmacological manipulation of the type 5 metabotropic glutamate (mGlu5) receptor alters various addiction related behaviors such as drug self-administration and the extinction and reinstatement of drug-seeking behavior. However, the effects of pharmacological modulation of mGlu5 receptors on brain reward function have not been widely investigated. We examined the effects of acute administration of positive and negative allosteric modulators (PAMs and NAMs, respectively) on brain reward function by assessing thresholds for intracranial self-stimulation (ICSS). In addition, when acute effects were observed, we examined changes in ICSS thresholds following repeated administration. Male Sprague-Dawley rats were implanted with bipolar electrodes into the medial forebrain bundle and trained to respond for ICSS, followed by assessment of effects of mGlu5 ligands on ICSS thresholds using a discrete trials current–intensity threshold determination procedure. Acute administration of the selective mGlu5 NAMs MTEP (0, 0.3, 1, or 3 mg/kg) and fenobam (0, 3, 10, or 30 mg/kg) dose-dependently increased ICSS thresholds (∼70% at the highest dose tested), suggesting a deficit in brain reward function. Acute administration of the mGlu5 PAMs CDPPB (0, 10, 30, and 60 mg/kg) or ADX47273 (0, 10, 30, and 60 mg/kg) was without effect at any dose tested. When administered once daily for five consecutive days, the development of tolerance to the ability of threshold-elevating doses of MTEP and fenobam to increase ICSS thresholds was observed. We conclude that mGlu5 PAMs and NAMs differentially affect brain reward function, and that tolerance to the ability of mGlu5 NAMs to reduce brain reward function develops with repeated administration. These brain reward deficits should be taken into consideration when interpreting acute effects of mGlu5 NAMs on drug self-administration, and repeated administration of these ligands may be an effective method to reduce these deficits. PMID

  12. Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs.

    PubMed

    Rook, Jerri M; Abe, Masahito; Cho, Hyekyung P; Nance, Kellie D; Luscombe, Vincent B; Adams, Jeffrey J; Dickerson, Jonathan W; Remke, Daniel H; Garcia-Barrantes, Pedro M; Engers, Darren W; Engers, Julie L; Chang, Sichen; Foster, Jarrett J; Blobaum, Anna L; Niswender, Colleen M; Jones, Carrie K; Conn, P Jeffrey; Lindsley, Craig W

    2017-01-10

    Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.

  13. Allosteric modulation of ATP-gated P2X receptor channels

    PubMed Central

    Coddou, Claudio; Stojilkovic, Stanko S.; Huidobro-Toro, J. Pablo

    2013-01-01

    Seven mammalian purinergic receptor subunits, denoted P2X1 to P2X7, and several spliced forms of these subunits have been cloned. When heterologously expressed, these cDNAs encode ATP-gated non-selective cation channels organized as trimers. All activated receptors produce cell depolarization and promote Ca2+ influx through their pores and indirectly by activating voltage-gated calcium channels. However, the biophysical and pharmacological properties of these receptors differ considerably, and the majority of these subunits are also capable of forming heterotrimers with other members of the P2X receptor family, which confers further different properties. These channels have three ATP binding domains, presumably located between neighboring subunits, and occupancy of at least two binding sites is needed for their activation. In addition to the orthosteric binding sites for ATP, these receptors have additional allosteric sites that modulate the agonist action at receptors, including sites for trace metals, protons, neurosteroids, reactive oxygen species and phosphoinositides. The allosteric regulation of P2X receptors is frequently receptor-specific and could be a useful tool to identify P2X members in native tissues and their roles in signaling. The focus of this review is on common and receptor-specific allosteric modulation of P2X receptors and the molecular base accounting for allosteric binding sites. PMID:21639805

  14. Development of Novel, CNS Penetrant Positive Allosteric Modulators for the Metabotropic Glutamate Receptor Subtype 1 (mGlu1), Based on an N-(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide Scaffold, That Potentiate Wild Type and Mutant mGlu1 Receptors Found in Schizophrenics.

    PubMed

    Garcia-Barrantes, Pedro M; Cho, Hyekyung P; Niswender, Colleen M; Byers, Frank W; Locuson, Charles W; Blobaum, Anna L; Xiang, Zixiu; Rook, Jerri M; Conn, P Jeffrey; Lindsley, Craig W

    2015-10-22

    The therapeutic potential of selective mGlu1 activation is vastly unexplored relative to the other group I mGlu receptor, mGlu5; therefore, our lab has focused considerable effort toward developing mGlu1 positive allosteric modulators (PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu1 PAMs based on an N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide scaffold provided 17e, a potent (mGlu1 EC50 = 31.8 nM) and highly CNS penetrant (brain to plasma ratio (Kp) of 1.02) mGlu1 PAM tool compound, that potentiated not only wild-type human mGlu1 but also mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenic patients. Moreover, both electrophysiological and in vivo studies indicate the mGlu1 ago-PAMs/PAMs do not possess the same epileptiform adverse effect liability as mGlu5 ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic window.

  15. Taurine allosterically modulates flunitrazepam binding to synaptic membranes.

    PubMed

    Quinn, M R; Miller, C L

    1992-09-01

    Taurine is hypothesized to exert its inhibitory neuromodulatory effects, in part, by interaction with the GABAA receptor. Although taurine displaces GABA agonist binding to synaptic membranes, its allosteric effects on the benzodiazepine recognition site of the GABAA receptor complex is unsettled. We determined the effects of taurine on [3H]flunitrazepam (Flu) binding to well-washed, frozen-thawed synaptic membranes prepared from rat cortex. Comparative binding studies were conducted at 37 degrees C and on ice (0-4 degrees C). At 37 degrees C taurine increased Flu binding in a concentration dependent way by interaction with a bicuculline sensitive site, similar to GABA. Taurine increased Flu binding by causing a decrease in KD. The maximal effectiveness of taurine on Flu binding could not be increased further by addition of GABA. In contrast, the maximal stimulation of Flu binding by GABA was decreased by addition of taurine to the level attained by taurine alone. These mixed agonist/antagonist effects of taurine are pharmacologically specific and qualify taurine as a partial GABA agonist in this type of allosteric interaction. However, taurine causes opposite effects on Flu binding when measured at 0-4 degrees C: taurine interacts with a bicuculline insensitive site to inhibit Flu binding by increasing the KD. Taurine inhibition of Flu binding is not overcome by increasing concentrations of GABA. Although the mechanism of taurine inhibition of Flu binding at 0-4 degrees C is unclear, it may be an indirect effect of taurine interaction with membrane phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

    PubMed

    Bernat, Viachaslau; Admas, Tizita Haimanot; Brox, Regine; Heinemann, Frank W; Tschammer, Nuska

    2014-11-21

    The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

  17. Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 3. Engineering plasma stability by discovery and optimization of isoindolinone analogs.

    PubMed

    Garcia-Barrantes, Pedro M; Cho, Hyekyung P; Blobaum, Anna L; Niswender, Colleen M; Conn, P Jeffrey; Lindsley, Craig W

    2016-04-15

    This Letter describes the further lead optimization of the VU0486321 series of mGlu1 positive allosteric modulators (PAMs), focused on addressing the recurrent issue of plasma instability of the phthalimide moiety. Here, we evaluated a number of phthalimide bioisosteres, and ultimately identified isoindolinones as the ideal replacement that effectively address plasma instability, while maintaining acceptable mGlu1 PAM potency, DMPK profile, CNS penetration and mGluR selectivity.

  18. 112 Gb/s transmission with a directly-modulated laser using FFT-based synthesis of orthogonal PAM and DMT signals.

    PubMed

    Ling, William A; Matsui, Yasuhiro; Daghighian, Henry M; Lyubomirsky, Ilya

    2015-07-27

    We report the experimental measurement of 112 Gb/s transmission back-to-back and through 12 km of S-SMF with a single directly-modulated laser (DML) using the novel modulation format Orthogonal PAM-DMT. This work demonstrates a record DML-based 112 Gb/s receiver sensitivity of -7.1 dBm at a BER of 10(-3), outperforming conventional PAM and DMT by approximately 2.5 dB.

  19. Quantitative Identification of Compound‐Dependent On‐Modules and Differential Allosteric Modules From Homologous Ischemic Networks

    PubMed Central

    Li, B; Liu, J; Zhang, YY; Wang, PQ; Yu, YN; Kang, RX; Wu, HL; Zhang, XX; Wang, YY

    2016-01-01

    Module‐based methods have made much progress in deconstructing biological networks. However, it is a great challenge to quantitatively compare the topological structural variations of modules (allosteric modules [AMs]) under different situations. A total of 23, 42, and 15 coexpression modules were identified in baicalin (BA), jasminoidin (JA), and ursodeoxycholic acid (UA) in a global anti‐ischemic mice network, respectively. Then, we integrated the methods of module‐based consensus ratio (MCR) and modified Zsummary module statistic to validate 12 BA, 22 JA, and 8 UA on‐modules based on comparing with vehicle. The MCRs for pairwise comparisons were 1.55% (BA vs. JA), 1.45% (BA vs. UA), and 1.27% (JA vs. UA), respectively. Five conserved allosteric modules (CAMs) and 17 unique allosteric modules (UAMs) were identified among these groups. In conclusion, module‐centric analysis may provide us a unique approach to understand multiple pharmacological mechanisms associated with differential phenotypes in the era of modular pharmacology. PMID:27758049

  20. Negative allosteric modulation of nicotinic acetylcholine receptors blocks nicotine self-administration in rats.

    PubMed

    Yoshimura, Ryan F; Hogenkamp, Derk J; Li, Wen Y; Tran, Minhtam B; Belluzzi, James D; Whittemore, Edward R; Leslie, Frances M; Gee, Kelvin W

    2007-12-01

    Drugs that antagonize nicotinic acetylcholine receptors (nAChRs) can be used to inhibit nicotine-induced behavior in both humans and animals. The aim of our experiments is to establish a proof-of-principle that antagonism of nAChRs by negative allosteric modulation can alter behavior in a relevant animal model of addiction, nicotine self-administration. We have identified a novel, negative allosteric modulator of nAChRs, UCI-30002 [N-(1,2,3,4-tetrahydro-1-naphthyl)-4-nitroaniline], with selectivity for the major neuronal nAChR subtypes over muscle-type nAChRs. After systemic administration, UCI-30002 significantly reduces nicotine self-administration in rats on both fixed ratio and progressive ratio schedules of reinforcement. The minimum effective dose that significantly alters nicotine self-administration corresponds to brain concentrations of UCI-30002 that produce at least 30% inhibition of the major neuronal nAChR subtypes measured in vitro. UCI-30002 has no effect on responding for food reinforcement in rats on either type of schedule, indicating that there is no effect on general responding or natural reward. UCI-30002 represents validation of the concept that negative allosteric modulators may have significant benefits as a strategy for treating nicotine addiction and encourages the development of subtype-selective modulators.

  1. The mGluR2 positive allosteric modulator, SAR218645, improves memory and attention deficits in translational models of cognitive symptoms associated with schizophrenia

    PubMed Central

    Griebel, Guy; Pichat, Philippe; Boulay, Denis; Naimoli, Vanessa; Potestio, Lisa; Featherstone, Robert; Sahni, Sukhveen; Defex, Henry; Desvignes, Christophe; Slowinski, Franck; Vigé, Xavier; Bergis, Olivier E.; Sher, Rosy; Kosley, Raymond; Kongsamut, Sathapana; Black, Mark D.; Varty, Geoffrey B.

    2016-01-01

    Normalization of altered glutamate neurotransmission through activation of the mGluR2 has emerged as a new approach to treat schizophrenia. These studies describe a potent brain penetrant mGluR2 positive allosteric modulator (PAM), SAR218645. The compound behaves as a selective PAM of mGluR2 in recombinant and native receptor expression systems, increasing the affinity of glutamate at mGluR2 as inferred by competition and GTPγ35S binding assays. SAR218645 augmented the mGluR2-mediated response to glutamate in a rat recombinant mGluR2 forced-coupled Ca2+ mobilization assay. SAR218645 potentiated mGluR2 agonist-induced contralateral turning. When SAR218645 was tested in models of the positive symptoms of schizophrenia, it reduced head twitch behavior induced by DOI, but it failed to inhibit conditioned avoidance and hyperactivity using pharmacological and transgenic models. Results from experiments in models of the cognitive symptoms associated with schizophrenia showed that SAR218645 improved MK-801-induced episodic memory deficits in rats and attenuated working memory impairment in NMDA Nr1neo−/− mice. The drug reversed disrupted latent inhibition and auditory-evoked potential in mice and rats, respectively, two endophenotypes of schizophrenia. This profile positions SAR218645 as a promising candidate for the treatment of cognitive symptoms of patients with schizophrenia, in particular those with abnormal attention and sensory gating abilities. PMID:27734956

  2. The mGluR2 positive allosteric modulator, SAR218645, improves memory and attention deficits in translational models of cognitive symptoms associated with schizophrenia.

    PubMed

    Griebel, Guy; Pichat, Philippe; Boulay, Denis; Naimoli, Vanessa; Potestio, Lisa; Featherstone, Robert; Sahni, Sukhveen; Defex, Henry; Desvignes, Christophe; Slowinski, Franck; Vigé, Xavier; Bergis, Olivier E; Sher, Rosy; Kosley, Raymond; Kongsamut, Sathapana; Black, Mark D; Varty, Geoffrey B

    2016-10-13

    Normalization of altered glutamate neurotransmission through activation of the mGluR2 has emerged as a new approach to treat schizophrenia. These studies describe a potent brain penetrant mGluR2 positive allosteric modulator (PAM), SAR218645. The compound behaves as a selective PAM of mGluR2 in recombinant and native receptor expression systems, increasing the affinity of glutamate at mGluR2 as inferred by competition and GTPγ(35)S binding assays. SAR218645 augmented the mGluR2-mediated response to glutamate in a rat recombinant mGluR2 forced-coupled Ca(2+) mobilization assay. SAR218645 potentiated mGluR2 agonist-induced contralateral turning. When SAR218645 was tested in models of the positive symptoms of schizophrenia, it reduced head twitch behavior induced by DOI, but it failed to inhibit conditioned avoidance and hyperactivity using pharmacological and transgenic models. Results from experiments in models of the cognitive symptoms associated with schizophrenia showed that SAR218645 improved MK-801-induced episodic memory deficits in rats and attenuated working memory impairment in NMDA Nr1(neo-/-) mice. The drug reversed disrupted latent inhibition and auditory-evoked potential in mice and rats, respectively, two endophenotypes of schizophrenia. This profile positions SAR218645 as a promising candidate for the treatment of cognitive symptoms of patients with schizophrenia, in particular those with abnormal attention and sensory gating abilities.

  3. A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

    PubMed Central

    Sun, Fen; Jin, Kunlin; Uteshev, Victor V.

    2013-01-01

    In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke. PMID:23951360

  4. mGlu5 positive allosteric modulation normalizes synaptic plasticity defects and motor phenotypes in a mouse model of Rett syndrome.

    PubMed

    Gogliotti, Rocco G; Senter, Rebecca K; Rook, Jerri M; Ghoshal, Ayan; Zamorano, Rocio; Malosh, Chrysa; Stauffer, Shaun R; Bridges, Thomas M; Bartolome, Jose M; Daniels, J Scott; Jones, Carrie K; Lindsley, Craig W; Conn, P Jeffrey; Niswender, Colleen M

    2016-05-15

    Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.

  5. Improving performance of mobile fronthaul architecture employing high order delta-sigma modulator with PAM-4 format.

    PubMed

    Li, Haibo; Hu, Rong; Yang, Qi; Luo, Ming; He, Zhixue; Jiang, Peng; Liu, Yongpiao; Li, Xiang; Yu, Shaohua

    2017-01-09

    An improved high-order delta-sigma modulator with multi-level quantizer is proposed to enable carrier aggregation of 4G-LTE signals in mobile fronthaul. Different from conventional delta-sigma modulation-based digital mobile fronthaul, a 2-bit quantizer is employed to reduce the quantization noise, which enabling the transmission via PAM-4 based IM-DD channel. Moreover, we employ the 4th-order high-pass filter (HPF) to replace the 1st-order HPF in the conventional delta-sigma modulator, resulting in a much better noise shaping performance. In the experiment, a PAM-4 based mobile fronthaul transmission of 32 aggregated 4G-LTE signals with a CPRI equivalent data rate of 39.32-Gb/s is demonstrated in a single-λ 10-Gb/s IM-DD channel. Significant improvement of 68% is achieved in the average EVM performance compared to the previous delta-sigma modulation-based digital mobile fronthaul.

  6. The Metabotropic Glutamate Receptor 4-Positive Allosteric Modulator VU0364770 Produces Efficacy Alone and in Combination with l-DOPA or an Adenosine 2A Antagonist in Preclinical Rodent Models of Parkinson's Disease

    PubMed Central

    Jones, Carrie K.; Bubser, Michael; Thompson, Analisa D.; Dickerson, Jonathan W.; Turle-Lorenzo, Nathalie; Amalric, Marianne; Blobaum, Anna L.; Bridges, Thomas M.; Morrison, Ryan D.; Jadhav, Satyawan; Engers, Darren W.; Italiano, Kimberly; Bode, Jacob; Daniels, J. Scott; Lindsley, Craig W.; Hopkins, Corey R.; Conn, P. Jeffrey

    2012-01-01

    Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu4), including N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu4 PAMs exhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu4 in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu4 PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with l-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of l-DOPA, suggesting that mGlu4 PAMs may provide l-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu4 PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either l-DOPA or A2A antagonists. PMID:22088953

  7. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  8. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  9. Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

    PubMed

    Kane, Lucas T; Costa, Blaise M

    2015-09-01

    The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders.

  10. Phytoplankton Photosynthetic Response During the Southern Ocean Iron Experiment Using Pulse Amplitude Modulated (PAM) Fluorescence

    NASA Astrophysics Data System (ADS)

    Peloquin, J. A.; Smith, W. O.

    2002-12-01

    Phytoplankton production in the Southern Ocean is controlled by complicated interactions of light, nutrients, and iron availability. In early 2002, the Southern Ocean Iron Experiment (SOFeX), was completed in the Southern Pacific (along ~ 170° W). Two iron enriched patches were created North and South of the Polar Front with initially distinct silicic acid concentrations, ~1 μM and ~60 μM, respectively. PAM was employed for measuring phytoplankton whole water (bulk) and single-celled photochemical efficiency of Photosystem II (Fv}/F{m). Bulk measurements of photochemical efficiency for PAM show increases as phytoplankton from iron-enriched patches are relieved from iron stress for both the North and South Patch. For single-celled analysis with PAM, a time course from the South Patch will be presented along with late North Patch data. For the Southern Patch, a distinct increase in Fv}/F{m with time was detected for all diatom genera analyzed and Fv}/F{m values continued to increase over the duration of analysis. For the late Northern Patch phytoplankton assemblage, bulk Fv}/F{m estimates were still elevated, ~0.5. However, the dominant species, Pseudonitzchia sp., exhibited lower photochemical efficiency (0.25-0.35), suggesting that it may have been experiencing iron or silica stress. In contrast, Fv}/F{m measurements of another dominant species, Phaeocystis antarctica, exhibited higher (0.5-0.6) values, suggesting that the colonial haptophyte was not significantly stressed at the time of measurement.

  11. Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

    PubMed Central

    Cook, A E; Mistry, S N; Gregory, K J; Furness, S G B; Sexton, P M; Scammells, P J; Conigrave, A D; Christopoulos, A; Leach, K

    2015-01-01

    BACKGROUND AND PURPOSE Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). CONCLUSIONS AND IMPLICATIONS The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses. PMID:25220431

  12. 2-Aminothienopyridazines as Novel Adenosine A1 Receptor Allosteric Modulators and Antagonists

    PubMed Central

    Ferguson, Gemma N.; Valant, Celine; Horne, James; Figler, Heidi; Flynn, Bernard L.; Linden, Joel; Chalmers, David K.; Sexton, Patrick M.; Christopoulos, Arthur; Scammells, Peter J.

    2008-01-01

    A pharmacophore-based screen identified 32 compounds including ethyl 5-amino-3-(4-tert-butylphenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylate (8) as a new allosteric modulator of the adenosine A1 receptor (A1AR). On the basis of this lead, various derivatives were prepared and evaluated for activity at the human A1AR. A number of the test compounds allosterically stabilized agonist-receptor-G protein ternary complexes in dissociation kinetic assays, but were found to be more potent as antagonists in subsequent functional assays of ERK1/2 phosphorylation. Additional experiments on the most potent antagonist, 13b, investigating A1AR-mediated [35S]GTPγS binding and [3H]CCPA equilibrium binding confirmed its antagonistic mode of action and also identified inverse agonism. This study has thus identified a new class of A1AR antagonists that can also recognize the receptor’s allosteric site with lower potency. PMID:18771255

  13. Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

    PubMed

    Admas, Tizita Haimanot; Bernat, Viachaslau; Heinrich, Markus R; Tschammer, Nuska

    2016-03-17

    The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

  14. Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two-state and operational models

    PubMed Central

    Roche, David; Gil, Debora; Giraldo, Jesús

    2013-01-01

    Two-state and operational models of both agonism and allosterism are compared to identify and characterize common pharmacological parameters. To account for the receptor-dependent basal response, constitutive receptor activity is considered in the operational models. By arranging two-state models as the fraction of active receptors and operational models as the fractional response relative to the maximum effect of the system, a one-by-one correspondence between parameters is found. The comparative analysis allows a better understanding of complex allosteric interactions. In particular, the inclusion of constitutive receptor activity in the operational model of allosterism allows the characterization of modulators able to lower the basal response of the system; that is, allosteric modulators with negative intrinsic efficacy. Theoretical simulations and overall goodness of fit of the models to simulated data suggest that it is feasible to apply the models to experimental data and constitute one step forward in receptor theory formalism. Linked Articles Another recent review on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476-5381.2012.02223.x And in this issue of BJP there is an article on a new allosteric modulator: Newman AS, Batis N, Grafton G, Caputo F, Brady CA, Lambert J, Peters JA, Gordon J, Brain KL, Powell AD and Barnes NM (2013). 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor. British Journal of Pharmacology 169: 1228–1238. doi: 10.1111/bph.12213 PMID:23647200

  15. Application Evaluation of Air-Sparging and Aerobic Bioremediation in PAM(Physical Aquifer Model) with Advanced and Integrated Module

    NASA Astrophysics Data System (ADS)

    Hong, U.; Ko, J.; Park, S.; Kim, Y.; Kwon, S.; Ha, J.; Lim, J.; Han, K.

    2010-12-01

    It is generally difficult for a single process to remediate contaminated soil and groundwater contaminated with various organic compounds such as total petroleum hydrocarbon (TPH), benzene, toluene, ethylbenzene, xylene (BTEX), chlorinated aliphatic hydrocarbons (CAHs) because those contaminants show different chemical properties in two phases (e.g. soil and groundwater). Therefore, it is necessary to design an in-situ remediation system which can remove various contaminants simultaneously. For the purpose, we constructed integrated well module which can apply several remediation process such as air sparging, soil vapor extraction, and bioventing. The advanced integrated module consisted of three main parts such as head, body, and end cap. First of all, head part has three 3.6-cm-diameter stainless lines and can simultaneously inject air or extract NAPL, respectively. Secondly, body part has two 10-cm-height screen intervals with 100-mesh stainless inserts for unsaturated and smear zone. Lastly, we constructed three different sizes of end caps for injection and extraction from a saturated zone. We assumed that the integrated module can play bioremediation, air sparging, cometabolic sparging, chemical oxidation. In this study, we examined application of air sparing and aerobic bioremediation of toluene in Physical Aquifer Model (PAM) with an integrated well module. During air sparging experiments, toluene concentration decreased by injection of air. In addition, we accomplished bioremediation experiment to evaluate removal of toluene by indigenous microbes in PAM with continuous air injection. From the two experiments result, we confirmed that air sparging and aerobic bioremediation processes can be simultaneously carried out by an intergrated well module.

  16. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  17. Identification of an allosteric modulator of the serotonin transporter with novel mechanism of action.

    PubMed

    Kortagere, Sandhya; Fontana, Andreia Cristina Karklin; Rose, Deja Renée; Mortensen, Ole Valente

    2013-09-01

    Serotonin transporters (SERTs) play an essential role in the termination and regulation of serotonin signaling in the brain. SERT is also the target of antidepressants and psychostimulants. Molecules with novel activities and modes of interaction with regard to SERT function are of great scientific and clinical interest. We explored structural regions outside the putative serotonin translocation pathway to identify potential binding sites for allosteric transporter modulators (ATMs). Mutational studies revealed a pocket of amino acids outside the orthosteric substrate binding sites located in the interface between extracellular loops 1 and 3 that when mutated affect transporter function. Using the structure of the bacterial transporter homolog leucine transporter as a template, we developed a structural model of SERT. We performed molecular dynamics simulations to further characterize the allosteric pocket that was identified by site-directed mutagenesis studies and employed this pocket in a virtual screen for small-molecule modulators of SERT function. In functional transport assays, we found that one of the identified molecules, ATM7, increased the reuptake of serotonin, possibly by facilitating the interaction of serotonin with transport-ready conformations of SERT when concentrations of serotonin were low and rate limiting. In addition, ATM7 potentiates 3,4-methylenedioxy-N-methylamphetamine (MDMA, "Ecstasy")-induced reversed transport by SERT. Taking advantage of a conformationally sensitive residue in transmembrane domain 6, we demonstrate that ATM7 mechanistically stabilizes an outward-facing conformation of SERT. Taken together these observations demonstrate that ATM7 acts through a novel mechanism that involves allosteric modulation of SERT function.

  18. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  19. Octahydropyrrolo[3,4-c]pyrrole negative allosteric modulators of mGlu1

    PubMed Central

    Manka, Jason T.; Rodriguez, Alice L.; Morrison, Ryan D.; Venable, Daryl F.; Cho, Hyekyung P.; Blobaum, Anna L.; Daniels, J. Scott; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2014-01-01

    Development of SAR in an octahydropyrrolo[3,4-c]pyrrole series of negative allosteric modulators of mGlu1 using a functional cell-based assay is described in this Letter. The octahydropyrrolo[3,4-c]pyrrole scaffold was chosen as an isosteric replacement for the piperazine ring found in the initial hit compound. Characterization of selected compounds in protein binding assays was used to identify the most promising analogs, which were then profiled in P450 inhibition assays in order to further assess the potential for drug-likeness within this series of compounds. PMID:23932792

  20. Positive Allosteric Modulators of Metabotropic Glutamate 2 Receptors in Schizophrenia Treatment

    PubMed Central

    Ellaithy, Amr; Younkin, Jason; Gonzalez-Maeso, Javier; Logothetis, Diomedes E.

    2015-01-01

    The last two decades have witnessed a rise in the “NMDA receptor hypofunction” hypothesis for schizophrenia, a devastating disorder that affects around 1% of the population worldwide. A variety of presynaptic, postsynaptic and regulatory proteins involved in glutamatergic signaling have thus been proposed as potential therapeutic targets. This Review focuses on positive allosteric modulation of metabotropic glutamate 2 receptors (mGlu2Rs) and discusses how recent preclinical epigenetic data may provide a molecular explanation for the discrepant results of clinical studies, further stimulating the field to exploit the promise of mGlu2R as a target for schizophrenia treatment. PMID:26148747

  1. Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

    SciTech Connect

    Jin,R.; Clark, S.; Weeks, A.; Dudman, J.; Gouaux, E.; Partin, K.

    2005-01-01

    Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

  2. Positive Allosteric Modulation of Insect Olfactory Receptor Function by ORco Agonists

    PubMed Central

    Tsitoura, Panagiota; Iatrou, Kostas

    2016-01-01

    Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function. PMID:28018173

  3. Experimental study of 112 Gb/s short reach transmission employing PAM formats and SiP intensity modulator at 1.3 μm.

    PubMed

    Chagnon, Mathieu; Osman, Mohamed; Poulin, Michel; Latrasse, Christine; Gagné, Jean-Frédéric; Painchaud, Yves; Paquet, Carl; Lessard, Stéphane; Plant, David

    2014-08-25

    We present a Silicon Photonic (SiP) intensity modulator operating at 1.3 μm with pulse amplitude modulation formats for short reach transmission employing a digital to analog converter for the RF signal generator, enabling pulse shaping and precompensation of the transmitter's frequency response. Details of the SiP Mach-Zehnder interfometer are presented. We study the system performance at various bit rates, PAM orders and propagation distances. To the best of our knowledge, we report the first demonstration of a 112 Gb/s transmission over 10 km of SMF fiber operating below pre-FEC BER threshold of 3.8 × 10(-3) employing PAM-8 at 37.4 Gbaud using a fully packaged SiP modulator. An analytical model for the Q-factor metric applicable for multilevel PAM-N signaling is derived and accurately experimentally verified in the case of Gaussian noise limited detection. System performance is experimentally investigated and it is demonstrated that PAM order selection can be optimally chosen as a function of the desired throughput. We demonstrate the ability of the proposed transmitter to exhibit software-defined transmission for short reach applications by selecting PAM order, symbol rate and pulse shape.

  4. Discovery of Potent Positive Allosteric Modulators of the α3β2 Nicotinic Acetylcholine Receptor by a Chemical Space Walk in ChEMBL

    PubMed Central

    2014-01-01

    While a plethora of ligands are known for the well studied α7 and α4β2 nicotinic acetylcholine receptor (nAChR), only very few ligands address the related α3β2 nAChR expressed in the central nervous system and at the neuromuscular junction. Starting with the public database ChEMBL organized in the chemical space of Molecular Quantum Numbers (MQN, a series of 42 integer value descriptors of molecular structure), a visual survey of nearest neighbors of the α7 nAChR partial agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU-282,987) pointed to N-(2-halobenzyl)-3-aminoquinuclidines as possible nAChR modulators. This simple “chemical space walk” was performed using a web-browser available at www.gdb.unibe.ch. Electrophysiological recordings revealed that these ligands represent a new and to date most potent class of positive allosteric modulators (PAMs) of the α3β2 nAChR, which also exert significant effects in vivo. The present discovery highlights the value of surveying chemical space neighbors of known drugs within public databases to uncover new pharmacology. PMID:24593915

  5. The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease.

    PubMed

    Jones, Carrie K; Bubser, Michael; Thompson, Analisa D; Dickerson, Jonathan W; Turle-Lorenzo, Nathalie; Amalric, Marianne; Blobaum, Anna L; Bridges, Thomas M; Morrison, Ryan D; Jadhav, Satyawan; Engers, Darren W; Italiano, Kimberly; Bode, Jacob; Daniels, J Scott; Lindsley, Craig W; Hopkins, Corey R; Conn, P Jeffrey; Niswender, Colleen M

    2012-02-01

    Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu₄), including N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu₄ PAMsexhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu₄ in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu₄ PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with L-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of L-DOPA, suggesting that mGlu₄ PAMs may provide L-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu₄ PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either L-DOPA or A2A antagonists.

  6. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  7. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

    PubMed Central

    Miao, Yinglong; Goldfeld, Dahlia Anne; Moo, Ee Von; Sexton, Patrick M.; Christopoulos, Arthur; McCammon, J. Andrew; Valant, Celine

    2016-01-01

    Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [3H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies. PMID:27601651

  8. Enhancement of social novelty discrimination by positive allosteric modulators at metabotropic glutamate 5 receptors: adolescent administration prevents adult-onset deficits induced by neonatal treatment with phencyclidine.

    PubMed

    Clifton, Nicholas E; Morisot, Nadège; Girardon, Sylvie; Millan, Mark J; Loiseau, Florence

    2013-02-01

    Metabotropic glutamate-5 receptors (mGluR5), which physically and functionally interact with N-methyl-D-Aspartate (NMDA) receptors, likewise control cognitive processes and have been proposed as targets for novel classes of antipsychotic agent. Since social cognition is impaired in schizophrenia and disrupted by NMDA receptor antagonists like dizocilpine, we evaluated its potential modulation by mGluR5. Acute administration (0.63-40 mg/kg) of the mGluR5 positive allosteric modulators (PAMs), 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and ADX47273, reversed a delay-induced impairment in social novelty discrimination (SND) in adult rats. The action of CDPPB was blocked by the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (2.5-10 mg/kg), and was also expressed upon microinjection into frontal cortex (0.63-10 μg/side), but not striatum. Supporting an interrelationship between mGluR5 and NMDA receptors, enhancement of SND by CDPPB was blocked by dizocilpine (0.08 mg/kg) while, reciprocally, dizocilpine-induced impairment in SND was attenuated by CDPPB (10 mg/kg). The SND deficit elicited by post-natal administration of phencyclidine (10 mg/kg, days 7-11) was reversed by CDPPB or ADX47273 in adults at week 8. This phencyclidine-induced impairment in cognition emerged in adult rats from week 7 on, and chronic, pre-symptomatic treatment of adolescent rats with CDPPB over weeks 5-6 (10 mg/kg per day) prevented the appearance of SND deficits in adults until at least week 13. In conclusion, as evaluated by a SND procedure, mGluR5 PAMs promote social cognition via actions expressed in interaction with NMDA receptors and exerted in frontal cortex. MGluR5 PAMs not only reverse but also (when given during adolescence) prevent the emergence of cognitive impairment associated with a developmental model of schizophrenia.

  9. Elucidation of direct competition and allosteric modulation of small-molecular-weight protein ligands using surface plasmon resonance methods.

    PubMed

    Huber, Walter; Sinopoli, Alessandro; Kohler, Josiane; Hug, Melanie; Ruf, Armin; Huber, Sylwia

    2015-08-01

    The present work introduces a surface plasmon resonance-based method for the discrimination of direct competition and allosteric effects that occur in ternary systems comprising a receptor protein and two small-molecular-weight ligands that bind to it. Fatty acid binding protein 4, fructose-1,6-bisphosphatase and human serum albumin were used as model receptor molecules to demonstrate the performance of the method. For each of the receptor molecules, pairs of ligand molecules were selected for which either direct competition or an allosteric effect had already been determined by other methods. The method of discrimination introduced here is based on the surface plasmon resonance responses observed at equilibrium when an immobilized receptor protein is brought into contact with binary mixtures of interacting ligands. These experimentally determined responses are compared with the responses calculated using a theoretical model that considers both direct competition and allosteric ligand interaction modes. This study demonstrates that the allosteric ternary complex model, which enables calculation of the fractional occupancy of the protein by each ligand in such ternary systems, is well suited for the theoretical calculation of these types of responses. For all of the ternary systems considered in this work, the experimental and calculated responses in the chosen concentration ratio range were identical within a five-σ confidence interval when the calculations considered the correct interaction mode of the ligands (direct competition or different types of allosteric regulation), and in case of allosteric modulation, also the correct strength of this effect. This study also demonstrates that the allosteric ternary complex model-based calculations are well suited to predict the ideal concentration ratio range or even single concentration ratios that can serve as hot spots for discrimination, and such hot spots can drastically reduce the numbers of measurements needed

  10. The sweet taste of true synergy: positive allosteric modulation of the human sweet taste receptor.

    PubMed

    Servant, Guy; Tachdjian, Catherine; Li, Xiaodong; Karanewsky, Donald S

    2011-11-01

    A diet low in carbohydrates helps to reduce the amount of ingested calories and to maintain a healthy weight. With this in mind, food and beverage companies have reformulated a large number of their products, replacing sugar or high fructose corn syrup with several different types of zero-calorie sweeteners to decrease or even totally eliminate their caloric content. A challenge remains, however, with the level of acceptance of some of these products in the market-place. Many consumers believe that zero-calorie sweeteners simply do not taste like sugar. A recent breakthrough reveals that positive allosteric modulators of the human sweet taste receptor, small molecules that enhance the receptor activity and sweetness perception, could be more effective than other reported taste enhancers at reducing calories in consumer products without compromising on the true taste of sugar. A unique mechanism of action at the receptor level could explain the robust synergy achieved with these new modulators.

  11. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

    PubMed Central

    Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  12. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    PubMed

    Wang, Wei; Hong, Jeong S; Rab, Andras; Sorscher, Eric J; Kirk, Kevin L

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  13. Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis

    PubMed Central

    Bai, Qifeng; Yao, Xiaojun

    2016-01-01

    Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1. PMID:26887338

  14. HPC Analysis of Multiple Binding Sites Communication and Allosteric Modulations in Drug Design: The HSP Case Study.

    PubMed

    Chiappori, Federica; Milanesi, Luciano; Merelli, Ivan

    2016-01-01

    Allostery is a long-range macromolecular mechanism of internal regulation, in which the binding of a ligand in an allosteric site induces distant conformational changes in a distant portion of the protein, modifying its activity. From the drug design point of view, this mechanism can be exploited to achieve important therapeutic effects, since ligands able to bind allosteric sites may be designed to regulate target proteins. Computational tools are a valid support in this sense, since they allow the characterization of allosteric communications within proteins, which are essential to design modulator ligands. While considering long-range interactions in macromolecules, the principal drug design tool available to researcher is molecular dynamics, and related applications, since it allows the evaluation of conformational changes of a protein bound to a ligand. In particular, all-atoms molecular dynamics is suitable to verify the internal mechanisms that orchestrate allosteric communications, in order to identify key residues and internal pathways that modify the protein behaviour. The problem is that these techniques are heavily time-consuming and computationally intensive, thus high performance computing systems, including parallel computing and GPU-accelerated computations, are necessary to achieve results in a reasonable time. In this review, we will discuss how it is possible to exploit in silico approaches to characterize allosteric modulations and long-range interactions within proteins, describing the case study of the Heat Shock Proteins, a class of chaperons regulated by stress conditions, which is particularly important since it is involved in many cancers and neurodegenerative diseases.

  15. Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis

    NASA Astrophysics Data System (ADS)

    Bai, Qifeng; Yao, Xiaojun

    2016-02-01

    Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.

  16. Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19

    PubMed Central

    Emmanouilidis, Leonidas; Schütz, Ulrike; Tripsianes, Konstantinos; Madl, Tobias; Radke, Juliane; Rucktäschel, Robert; Wilmanns, Matthias; Schliebs, Wolfgang; Erdmann, Ralf; Sattler, Michael

    2017-01-01

    The transport of peroxisomal membrane proteins (PMPs) requires the soluble PEX19 protein as chaperone and import receptor. Recognition of cargo PMPs by the C-terminal domain (CTD) of PEX19 is required for peroxisome biogenesis in vivo. Farnesylation at a C-terminal CaaX motif in PEX19 enhances the PMP interaction, but the underlying molecular mechanisms are unknown. Here, we report the NMR-derived structure of the farnesylated human PEX19 CTD, which reveals that the farnesyl moiety is buried in an internal hydrophobic cavity. This induces substantial conformational changes that allosterically reshape the PEX19 surface to form two hydrophobic pockets for the recognition of conserved aromatic/aliphatic side chains in PMPs. Mutations of PEX19 residues that either mediate farnesyl contacts or are directly involved in PMP recognition abolish cargo binding and cannot complement a ΔPEX19 phenotype in human Zellweger patient fibroblasts. Our results demonstrate an allosteric mechanism for the modulation of protein function by farnesylation. PMID:28281558

  17. Small Molecule Positive Allosteric Modulation of TRPV1 Activation by Vanilloids and Acidic pHS⃞

    PubMed Central

    Kaszas, Krisztian; Keller, Jason M.; Coddou, Claudio; Mishra, Santosh K.; Hoon, Mark A.; Stojilkovic, Stanko; Jacobson, Kenneth A.

    2012-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation. PMID:22005042

  18. Discovery of a Selective and CNS Penetrant Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 3 with Antidepressant and Anxiolytic Activity in Rodents

    PubMed Central

    Engers, Julie L.; Rodriguez, Alice L.; Konkol, Leah C.; Morrison, Ryan D.; Thompson, Analisa D.; Byers, Frank W.; Blobaum, Anna L.; Chang, Sichen; Venable, Daryl F.; Loch, Matthew T.; Niswender, Colleen M.; Daniels, J. Scott; Jones, Carrie K.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2016-01-01

    Previous preclinical work has demonstrated the therapeutic potential of antagonists of the group II metabotropic glutamate receptors (mGlus). Still, compounds that are selective for the individual group II mGlus (mGlu2 and mGlu3) have been scarce. There remains a need for such compounds with the balance of properties suitable for convenient use in a wide array of rodent behavioral studies. We describe here the discovery of a selective mGlu3 NAM 106 (VU0650786) suitable for in vivo work. Compound 106 is a member of a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-one compounds originally identified as a mGlu5 positive allosteric modulator (PAM) chemotype. Its suitability for use in rodent behavioral models has been established by extensive in vivo PK studies, and the behavioral experiments presented here with compound 106 represent the first examples in which an mGlu3 NAM has demonstrated efficacy in models where prior efficacy had previously been noted with nonselective group II antagonists. PMID:26335039

  19. Ligand-biased and probe-dependent modulation of chemokine receptor CXCR3 signaling by negative allosteric modulators.

    PubMed

    Bernat, Viachaslau; Brox, Regine; Heinrich, Markus R; Auberson, Yves P; Tschammer, Nuska

    2015-03-01

    Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

  20. Selective GABAA α5 Positive Allosteric Modulators Improve Cognitive Function in Aged Rats with Memory Impairment

    PubMed Central

    Koh, Ming Teng; Rosenzweig-Lipson, Sharon; Gallagher, Michela

    2012-01-01

    A condition of excess activity in the hippocampal formation is observed in the aging brain and in conditions that confer additional risk during aging for Alzheimer’s disease. Compounds that act as positive allosteric modulators at GABAA α5 receptors might be useful in targeting this condition because GABAA α5 receptors mediate tonic inhibition of principal neurons in the affected network. While agents to improve cognitive function in the past focused on inverse agonists, which are negative allosteric modulators at GABAA α5 receptors, research supporting that approach used only young animals and predated current evidence for excessive hippocampal activity in age-related conditions of cognitive impairment. Here, we used two compounds, Compound 44 [6,6-dimethyl-3-(3-hydroxypropyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one] and Compound 6 [methyl 3,5-diphenylpyridazine-4-carboxylate], with functional activity as potentiators of γ-aminobutyric acid at GABAA α5 receptors, to test their ability to improve hippocampal-dependent memory in aged rats with identified cognitive impairment. Improvement was obtained in aged rats across protocols differing in motivational and performance demands and across varying retention intervals. Significant memory improvement occurred after either intracereboventricular infusion with Compound 44 (100 μg) in a water maze task or systemic administration with Compound 6 (3 mg/kg) in a radial arm maze task. Furthermore, systemic administration improved behavioral performance at dosing shown to provide drug exposure in the brain and in vivo receptor occupancy in the hippocampus. These data suggest a novel approach to improve neural network function in clinical conditions of excess hippocampal activity. PMID:22732440

  1. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

    PubMed Central

    Dowal, Louisa; Sim, Derek S.; Dilks, James R.; Blair, Price; Beaudry, Sarah; Denker, Bradley M.; Koukos, Georgios; Kuliopulos, Athan; Flaumenhaft, Robert

    2011-01-01

    G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation. PMID:21282664

  2. [Escitalopram: a selective inhibitor and allosteric modulator of the serotonin transporter].

    PubMed

    Mnie-Filali, O; El Mansari, M; Scarna, H; Zimmer, L; Sánchez, C; Haddjeri, N

    2007-12-01

    , while chronic treatment with R-citalopram did not modify the basal proliferation rate in the dentate gyrus, it blocked the increase induced by escitalopram when coadministered. This suggests that neuronal adaptive changes, which are essential for antidepressant response, are rapidly induced by escitalopram but prevented by R-citalopram coadministration. The attenuating effect of R-citalopram was suggested to underlie the delayed recovery of 5-HT neuronal activity following long-term treatment with citalopram versus escitalopram. This is confirmed since a treatment with R-citalopram antagonized the recovery of firing observed in escitalopram-treated rats. The exact mechanism by which R-citalopram exerts its action is not yet fully defined; however, an allosteric interaction between the enantiomers and the 5-HT transporter (SERT) has been proposed. In this context, in vitro studies have revealed the existence of at least two binding sites on SERT: (1) a primary high-affinity binding site or orthosteric site that mediates the inhibition of 5-HT reuptake and (2) an allosteric low-affinity binding site that modulates the binding of ligands at the primary site. In presence of escitalopram alone, both the primary and the allosteric sites are occupied. Thus, escitalopram exerts a stabilizing effect on this association to SERT, resulting in an effective inhibition of 5-HT reuptake activity. On the other hand, in the presence of the two enantiomers, R-citalopram binds to the allosteric site and decreases the escitalopram action on SERT. Such an innovative mechanism of action can constitute a basis for development of new allosteric antidepressants that demonstrate higher efficacy and earlier onset of therapeutic effect.

  3. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

  4. Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels.

    PubMed

    Carswell, Casey L; Hénault, Camille M; Murlidaran, Sruthi; Therien, J P Daniel; Juranka, Peter F; Surujballi, Julian A; Brannigan, Grace; Baenziger, John E

    2015-09-01

    The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function.

  5. Allosteric Modulation of Hormone Release from Thyroxine and Corticosteroid-binding Globulins*

    PubMed Central

    Qi, Xiaoqiang; Loiseau, François; Chan, Wee Lee; Yan, Yahui; Wei, Zhenquan; Milroy, Lech-Gustav; Myers, Rebecca M.; Ley, Steven V.; Read, Randy J.; Carrell, Robin W.; Zhou, Aiwu

    2011-01-01

    The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr342 of the reactive loop and Tyr241 of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys243, which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg378. Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature. PMID:21325280

  6. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles.

    PubMed

    Bueno, Ana B; Showalter, Aaron D; Wainscott, David B; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over; Willard, Francis S; Sloop, Kyle W

    2016-05-13

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  7. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles*

    PubMed Central

    Showalter, Aaron D.; Wainscott, David B.; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over

    2016-01-01

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5′-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9–36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [3H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  8. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    PubMed

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  9. Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

    PubMed

    Rodgers, Thomas L; Townsend, Philip D; Burnell, David; Jones, Matthew L; Richards, Shane A; McLeish, Tom C B; Pohl, Ehmke; Wilson, Mark R; Cann, Martin J

    2013-09-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein "design space" that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  10. Allosteric Modulators of GABAB Receptors: Mechanism of Action and Therapeutic Perspective

    PubMed Central

    Pin, Jean-Philippe; Prézeau, Laurent

    2007-01-01

    γ-aminobutyric acid (GABA) plays important roles in the central nervous system, acting as a neurotransmitter on both ionotropic ligand-gated Cl--channels, and metabotropic G-protein coupled receptors (GPCRs). These two types of receptors called GABAA (and C) and GABAB are the targets of major therapeutic drugs such as the anxiolytic benzodiazepines, and antispastic drug baclofen (lioresal®), respectively. Although the multiplicity of GABAA receptors offer a number of possibilities to discover new and more selective drugs, the molecular characterization of the GABAB receptor revealed a unique, though complex, heterodimeric GPCR. High throughput screening strategies carried out in pharmaceutical industries, helped identifying new compounds positively modulating the activity of the GABAB receptor. These molecules, almost devoid of apparent activity when applied alone, greatly enhance both the potency and efficacy of GABAB agonists. As such, in contrast to baclofen that constantly activates the receptor everywhere in the brain, these positive allosteric modulators induce a large increase in GABAB-mediated responses only WHERE and WHEN physiologically needed. Such compounds are then well adapted to help GABA to activate its GABAB receptors, like benzodiazepines favor GABAA receptor activation. In this review, the way of action of these molecules will be presented in light of our actual knowledge of the activation mechanism of the GABAB receptor. We will then show that, as expected, these molecules have more pronounced in vivo responses and less side effects than pure agonists, offering new potential therapeutic applications for this new class of GABAB ligands. PMID:19305802

  11. Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

    PubMed

    Balsera, Beatriz; Mulet, José; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Ferrer-Montiel, Antonio; Hernández-Jiménez, José G; Estévez-Herrera, Judith; Borges, Ricardo; Freitas, Andiara E; López, Manuela G; García-López, M Teresa; González-Muñiz, Rosario; Pérez de Vega, María Jesús; Valor, Luis M; Svobodová, Lucie; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2014-10-30

    The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

  12. Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors*

    PubMed Central

    Bauer, Mark; Chicca, Andrea; Tamborrini, Marco; Eisen, David; Lerner, Raissa; Lutz, Beat; Poetz, Oliver; Pluschke, Gerd; Gertsch, Jürg

    2012-01-01

    The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [3H]CP55,940 and [3H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [3H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB1 receptors. Competition binding studies revealed Ki values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [35S]GTPγS binding, and CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling. PMID:22952224

  13. Lead optimization of the VU0486321 series of mGlu(1) PAMs. Part 2: SAR of alternative 3-methyl heterocycles and progress towards an in vivo tool.

    PubMed

    Garcia-Barrantes, Pedro M; Cho, Hyekyung P; Metts, Adam M; Blobaum, Anna L; Niswender, Colleen M; Conn, P Jeffrey; Lindsley, Craig W

    2016-02-01

    This Letter describes the further lead optimization of the VU0486321 series of mGlu1 positive allosteric modulators (PAMs), driven by recent genetic data linking loss of function GRM1 to schizophrenia. Steep and caveat-laden SAR plagues the series, but ultimately potent mGlu1 PAMs (EC50s ∼5 nM) have resulted with good DMPK properties (low intrinsic clearance, clean CYP profile, modest Fu) and CNS penetration (Kps 0.25-0.97), along with up to >450-fold selectivity versus mGlu4 and mGlu5.

  14. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  15. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

  16. [Pharmacological characteristics of drugs targeted on calcium-sensing receptor.-properties of cinacalcet hydrochloride as allosteric modulator].

    PubMed

    Nagano, Nobuo; Tsutsui, Takaaki

    2016-06-01

    Calcimimetics act as positive allosteric modulators of the calcium-sensing receptor (CaSR), thereby decreasing parathyroid hormone (PTH) secretion from the parathyroid glands. On the other hand, negative allosteric modulators of the CaSR with stimulatory effect on PTH secretion are termed calcilytics. The calcimimetic cinacalcet hydrochloride (cinacalcet) is the world's first allosteric modulator of G protein-coupled receptor to enter the clinical market. Cinacalcet just tunes the physiological effects of Ca(2+), an endogenous ligand, therefore, shows high selectivity and low side effects. Calcimimetics also increase cell surface CaSR expression by acting as pharmacological chaperones (pharmacoperones). It is considered that the cinacalcet-induced upper gastrointestinal problems are resulted from enhanced physiological responses to Ca(2+) and amino acids via increased sensitivity of digestive tract CaSR by cinacalcet. While clinical developments of calcilytics for osteoporosis were unfortunately halted or terminated due to paucity of efficacy, it is expected that calcilytics may be useful for the treatment of patients with activating CaSR mutations, asthma, and idiopathic pulmonary artery hypertension.

  17. Drugs modulate allosterically heme-Fe-recognition by human serum albumin and heme-fe-mediated reactivity.

    PubMed

    di Masi, Alessandra; Leboffe, Loris; Trezza, Viviana; Fanali, Gabriella; Coletta, Massimo; Fasano, Mauro; Ascenzi, Paolo

    2015-01-01

    Human serum albumin (HSA) represents an important determinant of plasma oncotic pressure and a relevant factor that modulates fluid distribution between the body compartments. Moreover, HSA (i) represents the depot and transporter of several compounds, both endogenous and exogenous, (ii) affects the pharmacokinetics of many drugs, (iii) regulates chemical modifications of some ligands, (iv) shows (pseudo-)enzymatic properties, (v) inactivates some toxic compounds, and (vi) displays anti-oxidant properties. HSA binding and (pseudo-)enzymatic properties are regulated competitively, allosterically, and by covalent modifications. While competitive inhibition of HSA binding properties is evident, allosteric mechanisms and covalent modifications affecting HSA reactivity are less clear. In several pathological conditions in which free heme-Fe levels increase, the buffering capacity of plasma hemopexin is overwhelmed and most of heme-Fe binds to the fatty acid site 1 of HSA. HSA-heme-Fe displays globin-like properties; in turn, heme-Fe modulates competitively and allosterically HSA binding and reactivity properties. Remarkably, heme-Fe-mediated HSA properties are time-dependent, representing a case for "chronosteric effects". Here, we review the drug-based modulation of (i) heme-Fe-recognition by HSA and (ii) heme-Fe-mediated reactivity.

  18. The membrane proximal region of the cannabinoid receptor CB1 N-terminus can allosterically modulate ligand affinity.

    PubMed

    Fay, Jonathan F; Farrens, David L

    2013-11-19

    The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

  19. Positive Allosteric Modulator of GABA Lowers BOLD Responses in the Cingulate Cortex

    PubMed Central

    Walter, Susanna A.; Forsgren, Mikael; Lundengård, Karin; Simon, Rozalyn; Torkildsen Nilsson, Maritha; Söderfeldt, Birgitta; Lundberg, Peter; Engström, Maria

    2016-01-01

    Knowledge about the neural underpinnings of the negative blood oxygen level dependent (BOLD) responses in functional magnetic resonance imaging (fMRI) is still limited. We hypothesized that pharmacological GABAergic modulation attenuates BOLD responses, and that blood concentrations of a positive allosteric modulator of GABA correlate inversely with BOLD responses in the cingulate cortex. We investigated whether or not pure task-related negative BOLD responses were co-localized with pharmacologically modulated BOLD responses. Twenty healthy adults received either 5 mg diazepam or placebo in a double blind, randomized design. During fMRI the subjects performed a working memory task. Results showed that BOLD responses in the cingulate cortex were inversely correlated with diazepam blood concentrations; that is, the higher the blood diazepam concentration, the lower the BOLD response. This inverse correlation was most pronounced in the pregenual anterior cingulate cortex and the anterior mid-cingulate cortex. For subjects with diazepam plasma concentration > 0.1 mg/L we observed negative BOLD responses with respect to fixation baseline. There was minor overlap between cingulate regions with task-related negative BOLD responses and regions where the BOLD responses were inversely correlated with diazepam concentration. We interpret that the inverse correlation between the BOLD response and diazepam was caused by GABA-related neural inhibition. Thus, this study supports the hypothesis that GABA attenuates BOLD responses in fMRI. The minimal overlap between task-related negative BOLD responses and responses attenuated by diazepam suggests that these responses might be caused by different mechanisms. PMID:26930498

  20. Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

    PubMed Central

    Shivnaraine, Rabindra V; Kelly, Brendan; Sankar, Krishana S; Redka, Dar'ya S; Han, Yi Rang; Huang, Fei; Elmslie, Gwendolynne; Pinto, Daniel; Li, Yuchong; Rocheleau, Jonathan V; Gradinaru, Claudiu C; Ellis, John; Wells, James W

    2016-01-01

    The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations. DOI: http://dx.doi.org/10.7554/eLife.11685.001 PMID:27151542

  1. A negative allosteric modulator demonstrates biased antagonism of the follicle stimulating hormone receptor

    PubMed Central

    Dias, James A.; Bonnet, Béatrice; Weaver, Barbara A.; Watts, Julie; Kluetzman, Kerri; Thomas, Richard M.; Poli, Sonia; Mutel, Vincent; Campo, Brice

    2015-01-01

    High quality gamete production in males and females requires the pituitary gonadotropin follicle stimulating hormone (FSH). In this report a novel chemical class of small molecule inhibitors of FSH receptor (FSHR) is described. ADX61623, a negative allosteric modulator (NAM), increased the affinity of interaction between 125I-hFSH and human FSHR (hFSHR) five fold. This form of FSHR occupied simultaneously by FSH and ADX61623 was inactive for cAMP and progesterone production in primary cultures of rat granulosa cells. In contrast, ADX61623 did not block estrogen production. This demonstrates for the first time, biased antagonism at the FSHR. To determine if ADX61623 blocked FSH induction of follicle development in vivo, a bioassay to measure follicular development and oocyte production in immature female rats was validated. ADX61623 was not completely effective in blocking FSH induced follicular development in vivo at doses up to 100 mg/kg as oocyte production and ovarian weight gain were only moderately reduced. These data illustrate that FSHR couples to multiple signaling pathways in vivo. Suppression of one pool of FSHR uncouples Gαs and cAMP production, and decreases progesterone production. Occupancy of another pool of FSHR sensitizes granulosa cells to FSH induced estradiol production. Therefore, ADX61623 is a useful tool to investigate further the mechanism of the FSHR signaling dichotomy. This may lead to a greater understanding of the signaling infrastructure which enables estrogen biosynthesis and may prove useful in treating estrogen dependent disease. PMID:21184806

  2. A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia

    PubMed Central

    Kalappa, Bopanna I; Sun, Fen; Johnson, Stephen R; Jin, Kunlin; Uteshev, Victor V

    2013-01-01

    Background and Purpose Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596. Experimental Approach An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats. Key Results Choline (20–200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg−1, s.c. and 1 mg·kg−1, i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg−1, i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments. Conclusions and Implications PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting

  3. Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2013-01-01

    Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity

  4. FUNCTIONAL ANALYSIS OF A NOVEL POSITIVE ALLOSTERIC MODULATOR OF AMPA RECEPTORS DERIVED FROM A STRUCTURE-BASED DRUG DESIGN STRATEGY

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Maclean, John K. F.; Partin, Kathryn M.; Jamieson, Craig

    2012-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors facilitate synaptic plasticity and can improve various forms of learning and memory. These modulators show promise as therapeutic agents for the treatment of neurological disorders such as schizophrenia, ADHD, and mental depression. Three classes of positive modulator, the benzamides, the thiadiazides, and the biarylsulfonamides differentially occupy a solvent accessible binding pocket at the interface between the two subunits that form the AMPA receptor ligand-binding pocket. Here, we describe the electrophysiological properties of a new chemotype derived from a structure-based drug design strategy (SBDD), which makes similar receptor interactions compared to previously reported classes of modulator. This pyrazole amide derivative, JAMI1001A, with a promising developability profile, efficaciously modulates AMPA receptor deactivation and desensitization of both flip and flop receptor isoforms. PMID:22735771

  5. The First Negative Allosteric Modulator for Dopamine D2 and D3 Receptors SB269652 May Lead to a New Generation of Antipsychotic Drugs.

    PubMed

    Rossi, Mario; Fasciani, Irene; Marampon, Francesco; Maggio, Roberto; Scarselli, Marco

    2017-03-06

    D2 and D3 dopamine receptors belong to the largest family of cell surface proteins in eukaryotes, the G protein-coupled receptors (GPCRs). Considering their crucial physiological functions and their relatively accessible cellular locations, GPCRs represent one of the most important classes of therapeutic targets. Until recently, the only strategy to develop drugs regulating GPCR activity was through the identification of compounds that directly acted on the orthosteric sites for endogenous ligands. However, many efforts have recently been made in order to identify small molecules that are able to interact with allosteric sites. These sites are less well-conserved; therefore, allosteric ligands have greater selectivity on the specific receptor. Strikingly, the use of allosteric modulators can provide specific advantages, such as an increased selectivity for GPCR subunits and the ability to introduce specific beneficial therapeutic effects without disrupting the integrity of complex physiologically-regulated networks. In 2010, our group unexpectedly found that SB269652, a compound supposed to interact with the orthosteric binding site of dopamine receptors, was actually a negative allosteric modulator of D2 and D3 receptor dimers, thus identifying the first allosteric small molecule acting on these important therapeutic targets. This review addresses the progresses in the understanding of the molecular mechanisms of interaction between the negative modulator SB269652 and D2 and D3 dopamine receptor monomers and dimers, and also the perspectives of developing new dopamine receptor allosteric drugs based on SB269652 as the leading compound.

  6. Dual allosteric modulation of opioid antinociceptive potency by α2A-adrenoceptors.

    PubMed

    Chabot-Doré, Anne-Julie; Millecamps, Magali; Naso, Lina; Devost, Dominic; Trieu, Phan; Piltonen, Marjo; Diatchenko, Luda; Fairbanks, Carolyn A; Wilcox, George L; Hébert, Terence E; Stone, Laura S

    2015-12-01

    Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using the hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10-70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2A-KO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management.

  7. Dual allosteric modulation of opioid antinociceptive potency by a2A-adrenoceptors

    PubMed Central

    Chabot-Doré, Anne-Julie; Millecamps, Magali; Naso, Lina; Devost, Dominic; Trieu, Phan; Piltonen, Marjo; Diatchenko, Luda; Fairbanks, Carolyn A.; Wilcox, George L.; Hébert, Terence E.; Stone, Laura S.

    2015-01-01

    Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10–70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2AKO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management. PMID:26254859

  8. Negative Allosteric Modulators of Metabotropic Glutamate Receptors Subtype 5 in Addiction: a Therapeutic Window

    PubMed Central

    2016-01-01

    Background: Abundant evidence at the anatomical, electrophysiological, and molecular levels implicates metabotropic glutamate receptor subtype 5 (mGluR5) in addiction. Consistently, the effects of a wide range of doses of different mGluR5 negative allosteric modulators (NAMs) have been tested in various animal models of addiction. Here, these studies were subjected to a systematic review to find out if mGluR5 NAMs have a therapeutic potential that can be translated to the clinic. Methods: Literature on consumption/self-administration and reinstatement of drug seeking as outcomes of interest published up to April 2015 was retrieved via PubMed. The review focused on the effects of systemic (i.p., i.v., s.c.) administration of the mGluR5 NAMs 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP) on paradigms with cocaine, ethanol, nicotine, and food in rats. Results: MTEP and MPEP were found to reduce self-administration of cocaine, ethanol, and nicotine at doses ≥1mg/kg and 2.5mg/kg, respectively. Dose-response relationship resembled a sigmoidal curve, with low doses not reaching statistical significance and high doses reliably inhibiting self-administration of drugs of abuse. Importantly, self-administration of cocaine, ethanol, and nicotine, but not food, was reduced by MTEP and MPEP in the dose range of 1 to 2mg/kg and 2.5 to 3.2mg/kg, respectively. This dose range corresponds to approximately 50% to 80% mGluR5 occupancy. Interestingly, the limited data found in mice and monkeys showed a similar therapeutic window. Conclusion: Altogether, this review suggests a therapeutic window for mGluR5 NAMs that can be translated to the treatment of substance-related and addictive disorders. PMID:26802568

  9. Discovery of molecular switches within the ADX-47273 mGlu5 PAM scaffold that modulate modes of pharmacology to afford potent mGlu5 NAMs, PAMs and partial antagonists.

    PubMed

    Lamb, Jeffrey P; Engers, Darren W; Niswender, Colleen M; Rodriguez, Alice L; Venable, Daryl F; Conn, P Jeffrey; Lindsley, Craig W

    2011-05-01

    This Letter describes a chemical lead optimization campaign directed at a weak mGlu(5) NAM discovered while developing SAR for the mGlu(5) PAM, ADX-47273. An iterative parallel synthesis effort discovered multiple, subtle molecular switches that afford potent mGlu(5) NAMs, mGlu(5) PAMs as well as mGlu(5) partial antagonists.

  10. Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

    PubMed Central

    Miller, Laurence J.

    2010-01-01

    It is useful to consider seven transmembrane receptors (7TMRs) as disordered proteins able to allosterically respond to a number of binding partners. Considering 7TMRs as allosteric systems, affinity and efficacy can be thought of in terms of energy flow between a modulator, conduit (the receptor protein), and a number of guests. These guests can be other molecules, receptors, membrane-bound proteins, or signaling proteins in the cytosol. These vectorial flows of energy can yield standard canonical guest allostery (allosteric modification of drug effect), effects along the plane of the cell membrane (receptor oligomerization), or effects directed into the cytosol (differential signaling as functional selectivity). This review discusses these apparently diverse pharmacological effects in terms of molecular dynamics and protein ensemble theory, which tends to unify 7TMR behavior toward cells. Special consideration will be given to functional selectivity (biased agonism and biased antagonism) in terms of mechanism of action and potential therapeutic application. The explosion of technology that has enabled observation of diverse 7TMR behavior has also shown how drugs can have multiple (pluridimensional) efficacies and how this can cause paradoxical drug classification and nomenclatures. PMID:20392808

  11. Positive allosteric modulation of A1 adenosine receptors as a novel and promising therapeutic strategy for anxiety.

    PubMed

    Vincenzi, Fabrizio; Ravani, Annalisa; Pasquini, Silvia; Merighi, Stefania; Gessi, Stefania; Romagnoli, Romeo; Baraldi, Pier Giovanni; Borea, Pier Andrea; Varani, Katia

    2016-12-01

    Activation of A1 adenosine receptors (ARs) has been associated with anxiolytic-like effects in different behavioral tests, but development of A1AR agonists for therapeutic use has been hampered, most likely due to the presence of side effects. With the aim to identify a safer approach for the treatment of anxiety, we investigated, in mice, the anxiolytic-like properties of a novel A1AR positive allosteric modulator, TRR469. Acute administration of TRR469 (0.3-3 mg/kg) resulted in robust anxiolytic-like effects in the elevated plus maze, the dark/light box, the open field and the marble burying tests. The magnitude of the anxiolytic action of TRR469 was comparable to that obtained with benzodiazepine diazepam (1 mg/kg). The use of the A1AR antagonist DPCPX (3 mg/kg) suggested that the effects of TRR469 were mediated by this receptor subtype. In contrast to diazepam, the novel positive allosteric modulator did not potentiate the sedative effect of ethanol (3.5 g/kg) evaluated by the loss of righting reflex. While diazepam produced motor coordination impairment in the rotarod test, this effect being enhanced by the presence of ethanol (1.5 g/kg), TRR469 did not elicit locomotor disturbances either when administered alone or in the presence of ethanol. In vitro, TRR469 was able to increase the number of A1AR recognizable by the agonist radioligand [(3)H]-CCPA in mouse brain regions involved in emotional processes. TRR469 markedly increased the affinity of the agonist CCPA, suggesting the capability, in vivo, to increase the affinity of endogenous adenosine. Taken together, these findings indicate that the positive allosteric modulation of A1AR may represent a promising approach for the treatment of anxiety-related disorders.

  12. Anticonvulsant effects of structurally diverse GABA(B) positive allosteric modulators in the DBA/2J audiogenic seizure test: Comparison to baclofen and utility as a pharmacodynamic screening model.

    PubMed

    Brown, Jordan W; Moeller, Achim; Schmidt, Martin; Turner, Sean C; Nimmrich, Volker; Ma, Junli; Rueter, Lynne E; van der Kam, Elizabeth; Zhang, Min

    2016-02-01

    The GABA(B) receptor has been indicated as a promising target for multiple CNS-related disorders. Baclofen, a prototypical orthosteric agonist, is used clinically for the treatment of spastic movement disorders, but is associated with unwanted side-effects, such as sedation and motor impairment. Positive allosteric modulators (PAM), which bind to a topographically-distinct site apart from the orthosteric binding pocket, may provide an improved side-effect profile while maintaining baclofen-like efficacy. GABA, the major inhibitory neurotransmitter in the CNS, plays an important role in the etiology and treatment of seizure disorders. Baclofen is known to produce anticonvulsant effects in the DBA/2J mouse audiogenic seizure test (AGS), suggesting it may be a suitable assay for assessing pharmacodynamic effects. Little is known about the effects of GABA(B) PAMs, however. The studies presented here sought to investigate the AGS test as a pharmacodynamic (PD) screening model for GABA(B) PAMs by comparing the profile of structurally diverse PAMs to baclofen. GS39783, rac-BHFF, CMPPE, A-1295120 (N-(3-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide), and A-1474713 (N-(3-(4-(4-chlorobenzyl)-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide) all produced robust, dose-dependent anticonvulsant effects; a similar profile was observed with baclofen. Pre-treatment with the GABA(B) antagonist SCH50911 completely blocked the anticonvulsant effects of baclofen and CMPPE in the AGS test, indicating such effects are likely mediated by the GABA(B) receptor. In addition to the standard anticonvulsant endpoint of the AGS test, video tracking software was employed to assess potential drug-induced motor side-effects during the acclimation period of the test. This analysis was sensitive to detecting drug-induced changes in total distance traveled, which was used to establish a therapeutic index (TI = hypoactivity

  13. Positional isomers of bispyridine benzene derivatives induce efficacy changes on mGlu5 negative allosteric modulation.

    PubMed

    Gómez-Santacana, Xavier; Dalton, James A R; Rovira, Xavier; Pin, Jean Philippe; Goudet, Cyril; Gorostiza, Pau; Giraldo, Jesús; Llebaria, Amadeu

    2017-02-15

    Modulation of metabotropic glutamate receptor 5 (mGlu5) with partial allosteric antagonists has received increased interest due to their favourable in vivo activity profiles compared to the unfavourable side-effects of full inverse agonists. Here we report on a series of bispyridine benzene derivatives with a functional molecular switch affecting antagonistic efficacy, shifting from inverse agonism to partial antagonism with only a single change in the substitution pattern of the benzene ring. These efficacy changes are explained through computational docking, revealing two different receptor conformations of different energetic stability and different positional isomer binding preferences.

  14. Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation

    PubMed Central

    Horenstein, Nicole A.; Papke, Roger L.; Kulkarni, Abhijit R.; Chaturbhuj, Ganesh U.; Stokes, Clare; Manther, Khan; Thakur, Ganesh A.

    2016-01-01

    The α7 nicotinic acetylcholine receptors (nAChRs) are uniquely sensitive to selective positive allosteric modulators (PAMs), which increase the efficiency of channel activation to a level greater than that of other nAChRs. Although PAMs must work in concert with “orthosteric” agonists, compounds such as GAT107 ((3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) have the combined properties of agonists and PAMs (ago-PAM) and produce very effective channel activation (direct allosteric activation (DAA)) by operating at two distinct sites in the absence of added agonist. One site is likely to be the same transmembrane site where PAMs like PNU-120596 function. We show that the other site, required for direct activation, is likely to be solvent-accessible at the extracellular domain vestibule. We identify key attributes of molecules in this family that are able to act at the DAA site through variation at the aryl ring substituent of the tetrahydroquinoline ring system and with two different classes of competitive antagonists of DAA. Analyses of molecular features of effective allosteric agonists allow us to propose a binding model for the DAA site, featuring a largely non-polar pocket accessed from the extracellular vestibule with an important role for Asp-101. This hypothesis is supported with data from site-directed mutants. Future refinement of the model and the characterization of specific GAT107 analogs will allow us to define critical structural elements that can be mapped onto the receptor surface for an improved understanding of this novel way to target α7 nAChR therapeutically. PMID:26742843

  15. Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 1: SAR of modifications to the central aryl core.

    PubMed

    Garcia-Barrantes, Pedro M; Cho, Hyekyung P; Blobaum, Anna L; Niswender, Colleen M; Conn, P Jeffrey; Lindsley, Craig W

    2015-11-15

    This Letter describes the lead optimization of the VU0486321 series of mGlu1 positive allosteric modulators (PAMs). While first generation PAMs from Roche were reported in the late 1990s, little effort has focused on the development of mGlu1 PAMs since. New genetic data linking loss-of-function mutant mGlu1 receptors to schizophrenia, bipolar disorder and other neuropsychiatric disorders has rekindled interest in the target, but the ideal in vivo probe, for example, with good PK, brain penetration and low plasma protein binding, for robust target validation has been lacking. Here we describe the first modifications to the central aryl core of the VU0486321 series, where robust SAR was noted. Moreover, structural variants were identified that imparted selectivity (up to >793-fold) versus mGlu4.

  16. PAM stack test utility

    SciTech Connect

    Grondona, Mark A.

    2007-08-22

    The pamtest utility calls the normal PAM hooks using a service and username supplied on the command line. This allows an administratory to test any one of many configured PAM stacks as any existing user on the machine.

  17. Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator.

    PubMed

    Font, Joan; López-Cano, Marc; Notartomaso, Serena; Scarselli, Pamela; Di Pietro, Paola; Bresolí-Obach, Roger; Battaglia, Giuseppe; Malhaire, Fanny; Rovira, Xavier; Catena, Juanlo; Giraldo, Jesús; Pin, Jean-Philippe; Fernández-Dueñas, Víctor; Goudet, Cyril; Nonell, Santi; Nicoletti, Ferdinando; Llebaria, Amadeu; Ciruela, Francisco

    2017-04-11

    Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.

  18. Efficacy of selective PDE4D negative allosteric modulators in the object retrieval task in female cynomolgus monkeys (Macaca fascicularis).

    PubMed

    Sutcliffe, Jane S; Beaumont, Vahri; Watson, James M; Chew, Chang Sing; Beconi, Maria; Hutcheson, Daniel M; Dominguez, Celia; Munoz-Sanjuan, Ignacio

    2014-01-01

    Cyclic adenosine monophosphate (cAMP) signalling plays an important role in synaptic plasticity and information processing in the hippocampal and basal ganglia systems. The augmentation of cAMP signalling through the selective inhibition of phosphodiesterases represents a viable strategy to treat disorders associated with dysfunction of these circuits. The phosphodiesterase (PDE) type 4 inhibitor rolipram has shown significant pro-cognitive effects in neurological disease models, both in rodents and primates. However, competitive non-isoform selective PDE4 inhibitors have a low therapeutic index which has stalled their clinical development. Here, we demonstrate the pro-cognitive effects of selective negative allosteric modulators (NAMs) of PDE4D, D159687 and D159797 in female Cynomolgous macaques, in the object retrieval detour task. The efficacy displayed by these NAMs in a primate cognitive task which engages the corticostriatal circuitry, together with their suitable pharmacokinetic properties and safety profiles, suggests that clinical development of these allosteric modulators should be considered for the treatment of a variety of brain disorders associated with cognitive decline.

  19. Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

    PubMed

    Chambon, Caroline; Jatzke, Claudia; Wegener, Nico; Gravius, Andreas; Danysz, Wojciech

    2012-12-15

    Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10μM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease.

  20. Efficacy of Selective PDE4D Negative Allosteric Modulators in the Object Retrieval Task in Female Cynomolgus Monkeys (Macaca fascicularis)

    PubMed Central

    Sutcliffe, Jane S.; Beaumont, Vahri; Watson, James M.; Chew, Chang Sing; Beconi, Maria; Hutcheson, Daniel M.; Dominguez, Celia; Munoz-Sanjuan, Ignacio

    2014-01-01

    Cyclic adenosine monophosphate (cAMP) signalling plays an important role in synaptic plasticity and information processing in the hippocampal and basal ganglia systems. The augmentation of cAMP signalling through the selective inhibition of phosphodiesterases represents a viable strategy to treat disorders associated with dysfunction of these circuits. The phosphodiesterase (PDE) type 4 inhibitor rolipram has shown significant pro-cognitive effects in neurological disease models, both in rodents and primates. However, competitive non-isoform selective PDE4 inhibitors have a low therapeutic index which has stalled their clinical development. Here, we demonstrate the pro-cognitive effects of selective negative allosteric modulators (NAMs) of PDE4D, D159687 and D159797 in female Cynomolgous macaques, in the object retrieval detour task. The efficacy displayed by these NAMs in a primate cognitive task which engages the corticostriatal circuitry, together with their suitable pharmacokinetic properties and safety profiles, suggests that clinical development of these allosteric modulators should be considered for the treatment of a variety of brain disorders associated with cognitive decline. PMID:25050979

  1. Structural and Functional Analysis of Two New Positive Allosteric Modulators of GluA2 Desensitization and Deactivation

    PubMed Central

    Timm, David E.; Benveniste, Morris; Weeks, Autumn M.; Nisenbaum, Eric S.

    2011-01-01

    At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1–3′,2′)1,3-oxazino(6′,5′-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously. PMID:21543522

  2. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  3. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

    NASA Astrophysics Data System (ADS)

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-10-01

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

  4. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

    PubMed Central

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-01-01

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands. PMID:26482099

  5. The GABAB Positive Allosteric Modulator, ADX71441 Attenuates Alcohol Self-Administration and Relapse to Alcohol Seeking in Rats.

    PubMed

    Augier, Eric; Dulman, Russell S; Damadzic, Ruslan; Pilling, Andrew; Hamilton, J Paul; Heilig, Markus

    2017-03-15

    GABAergic signaling is involved in modulating the reinforcing properties of alcohol, and GABAB receptors have been proposed as a potential target for clinical treatment of alcoholism. The orthosteric GABAB receptor agonist baclofen has been shown to suppress operant self-administration of alcohol in animals and alcohol use in alcohol dependent patients, but its utility is limited by a narrow therapeutic index. We tested the effects of ADX71441, a novel GABAB receptor positive allosteric modulator on alcohol-related behaviors in rats. We first assessed the effects of ADX71441 (1, 3, 10 and 30 mg/kg, I.P.) on both non-dependent and dependent male Wistar rats trained to self-administer 20% alcohol. We then determined the effects of ADX71441 on stress-induced as well as cue-induced relapse-like behavior. Finally, we sought to identify the brain regions through which ADX71441 may act to prevent relapse-like behavior by mapping the neuronal activation induced by stress-induced reinstatement of alcohol-seeking using c-Fos immunohistochemistry. ADX71441 dose-dependently decreased alcohol self-administration of both dependent and non-dependent animals, but its potency was higher in alcohol-dependent rats. Furthermore, both cue- and stress-induced alcohol seeking were blocked by the GABAB receptor positive allosteric modulator. Finally, pretreatment with 3 mg/kg of ADX71441 before stress-induced reinstatement significantly decreased c-Fos expression in a network of brain regions implicated in stress induced relapse, comprising the nucleus accumbens shell, the dorsal raphe nucleus and the medial prefrontal cortex. Our findings support a causal role of GABAB receptors in alcohol reinforcement and relapse to alcohol seeking. These effects are observed in the absence of significant sedative side effects. Jointly, these observations indicate that GABAB receptor positive allosteric modulators merit being tested clinically for the treatment of alcoholism. Our data also point to

  6. Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to its Heparin-Binding Site

    PubMed Central

    Karuturi, Rajesh; Al-Horani, Rami A.; Mehta, Shrenik C.; Gailani, David; Desai, Umesh R.

    2013-01-01

    To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs. PMID:23451707

  7. Positive Allosteric Modulation of mGluR5 Accelerates Extinction Learning but Not Relearning Following Methamphetamine Self-Administration

    PubMed Central

    Kufahl, Peter R.; Hood, Lauren E.; Nemirovsky, Natali E.; Barabas, Piroska; Halstengard, Casey; Villa, Angel; Moore, Elisabeth; Watterson, Lucas R.; Olive, M. Foster

    2012-01-01

    Recent studies have implicated glutamate neurotransmission as an important substrate for the extinction of conditioned behaviors, including responding for drug reinforcement. Positive allosteric modulation of the type-5 metabotropic glutamate receptor (mGluR5) in particular has emerged as a treatment strategy for the enhancement of extinction of drug-motivated behaviors. Here, we investigated the effects of the mGluR5 positive allosteric modulator CDPPB, a compound known for its cognitive enhancing effects in rodents, on extinction learning in rats with different histories of methamphetamine (METH) training. Rats were trained to self-administer METH under two conditions: 16 daily sessions of short access (90 min/day, ShA), or eight daily sessions of short access followed by eight sessions of long access (6 h/day, LgA). Control rats self-administered sucrose pellets in daily 30 min sessions. Next, rats were administered vehicle or 30 mg/kg CDPPB prior to seven consecutive daily extinction sessions, subjected to additional extinction sessions to re-establish a post-treatment baseline, and then tested for reinstatement of behavior in the presence of METH- or sucrose-paired cues. Rats were then subjected to a second series of extinction sessions, preceded by vehicle or 30 mg/kg CDPPB, and an additional test for cue-triggered reinstatement. CDPPB treatment resulted in a more rapid extinction of responding on the active lever, especially in the early sessions of the first extinction sequence. However, treatment effects were minimal during subsequent cue reinstatement tests and non-existent during the second series of extinction sessions. Rats with histories of ShA, LgA, and sucrose training expressed similar behavioral sensitivities to CDPPB, with LgA rats demonstrating a modestly higher treatment effect. Positive allosteric modulation of mGluR5 may therefore have some beneficial effects on efforts to facilitate extinction learning and reduce methamphetamine

  8. Targeting Selective Activation of M1 for the Treatment of Alzheimer’s Disease: Further Chemical Optimization and Pharmacological Characterization of the M1 Positive Allosteric Modulator ML169

    PubMed Central

    2012-01-01

    The M1 muscarinic acetylcholine receptor is thought to play an important role in memory and cognition, making it a potential target for the treatment of Alzheimer’s disease (AD) and schizophrenia. Moreover, M1 interacts with BACE1 and regulates its proteosomal degradation, suggesting selective M1 activation could afford both palliative cognitive benefit as well as disease modification in AD. A key challenge in targeting the muscarinic acetylcholine receptors is achieving mAChR subtype selectivity. Our lab has previously reported the M1 selective positive allosteric modulator ML169. Herein we describe our efforts to further optimize this lead compound by preparing analogue libraries and probing novel scaffolds. We were able to identify several analogues that possessed submicromolar potency, with our best example displaying an EC50 of 310 nM. The new compounds maintained complete selectivity for the M1 receptor over the other subtypes (M2–M5), displayed improved DMPK profiles, and potentiated the carbachol (CCh)-induced excitation in striatal MSNs. Selected analogues were able to potentiate CCh-mediated nonamyloidogenic APPsα release, further strengthening the concept that M1 PAMs may afford a disease-modifying role in the treatment of AD. PMID:23173069

  9. Recent Advances in the Design and Development of Novel Negative Allosteric Modulators of mGlu5

    PubMed Central

    2011-01-01

    Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have remained attractive to researchers as potential therapies for a number of central nervous system related diseases, including anxiety, pain, gastresophageal reflux disease (GERD), addiction, Parkinson’s disease (PD), and fragile X syndrome (FXS). In addition to the many publications with supportive preclinical data with key tool molecules, recent positive reports from the clinic have bolstered the confidence in this approach. During the 2 year time span from 2009 through 2010, a number of new mGlu5 NAM chemotypes have been disclosed and discussed in the primary and patent literature. A summary of several efforts representing many diverse chemotypes are presented here, along with a discussion of representative structure–activity relationships (SAR) and synthetic approaches to the templates where possible. PMID:21927649

  10. Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

    PubMed Central

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

  11. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    PubMed

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  12. The positive allosteric modulator of the GABA(B) receptor, rac-BHFF, suppresses alcohol self-administration.

    PubMed

    Maccioni, Paola; Thomas, Andrew W; Carai, Mauro A M; Gessa, Gian Luigi; Malherbe, Pari; Colombo, Giancarlo

    2010-06-01

    The present study was designed to extend to the newly synthesized rac-BHFF [(R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one] the investigation on the capacity of positive allosteric modulators of the GABA(B) receptor to reduce alcohol self-administration in rats. To this end, selectively bred Sardinian alcohol-preferring (sP) rats were initially trained to respond on a lever [on a fixed ratio 4 (FR4) schedule of reinforcement] to orally self-administer alcohol (15%, v/v) or sucrose (0.7%, w/v) in daily 30-min sessions. Once responding reached stable levels, the effect of rac-BHFF (0, 50, 100, and 200mg/kg; i.g.) on responding for alcohol and sucrose was determined. Pretreatment with rac-BHFF produced a dose-dependent suppression in responding for alcohol; reduction in the total number of responses for alcohol, in comparison to vehicle-treated rats, averaged approximately 30%, 65%, and 90% in 50, 100, and 200mg/kg rac-BHFF-treated rats, respectively. Pretreatment with 200mg/kg rac-BHFF markedly increased the latency to the first response on the alcohol lever. The effect of pretreatment with rac-BHFF on alcohol self-administration was highly specific, since (a) responding for sucrose was reduced (to approximately 50%, in comparison to vehicle-treated rats) only by pretreatment with 200mg/kg rac-BHFF, and (b) latency to the first response on the sucrose lever was completely unaltered by any rac-BHFF dose. Treatment with rac-BHFF did not alter spontaneous locomotor activity in an independent group of sP rats. The present data constitute a further piece of evidence on the capacity of positive allosteric modulators of the GABA(B) receptor to reduce alcohol's reinforcing properties in rats.

  13. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo.

    PubMed

    Timmermann, Daniel B; Grønlien, Jens Halvard; Kohlhaas, Kathy L; Nielsen, Elsebet Ø; Dam, Eva; Jørgensen, Tino D; Ahring, Philip K; Peters, Dan; Holst, Dorte; Christensen, Jeppe K; Chrsitensen, Jeppe K; Malysz, John; Briggs, Clark A; Gopalakrishnan, Murali; Olsen, Gunnar M

    2007-10-01

    Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

  14. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

    PubMed Central

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  15. Modulation of γ-secretase specificity using small molecule allosteric inhibitors

    PubMed Central

    Shelton, Christopher C.; Zhu, Lei; Chau, Deming; Yang, Li; Wang, Rong; Djaballah, Hakim; Zheng, Hui; Li, Yue-Ming

    2009-01-01

    γ-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of γ-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of γ-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit γ-secretase-mediated production of Aβ42 over other cleavage activities. These coumarin dimer-based compounds interact with γ-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of γ-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Aβ42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which γ-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of γ-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents. PMID:19906985

  16. Arsenic toxicity in the water weed Wolffia arrhiza measured using Pulse Amplitude Modulation Fluorometry (PAM) measurements of photosynthesis.

    PubMed

    Ritchie, Raymond J; Mekjinda, Nutsara

    2016-10-01

    Accumulation of arsenic in plants is a serious South-east Asian environmental problem. Photosynthesis in the small aquatic angiosperm Wolffia arrhiza is very sensitive to arsenic toxicity, particularly in water below pH 7 where arsenite (As (OH)3) (AsIII) is the dominant form; at pH >7 AsO4(2-) (As(V) predominates). A blue-diode PAM (Pulse Amplitude Fluorometer) machine was used to monitor photosynthesis in Wolffia. Maximum gross photosynthesis (Pgmax) and not maximum yield (Ymax) is the most reliable indicator of arsenic toxicity. The toxicity of arsenite As(III) and arsenate (H2AsO4(2-)) As(V) vary with pH. As(V) was less toxic than As(III) at both pH 5 and pH 8 but both forms of arsenic were toxic (>90% inhibition) at below 0.1molm(-3) when incubated in arsenic for 24h. Arsenite toxicity was apparent after 1h based on Pgmax and gradually increased over 7h but there was no apparent effect on Ymax or photosynthetic efficiency (α0).

  17. Allosteric modulation of the effect of escitalopram, paroxetine and fluoxetine: in-vitro and in-vivo studies.

    PubMed

    Mansari, Mostafa El; Wiborg, Ove; Mnie-Filali, Ouissame; Benturquia, Nadia; Sánchez, Connie; Haddjeri, Nasser

    2007-02-01

    Clinical and preclinical studies have shown that the effect of citalopram on serotonin (5-HT) reuptake inhibition and its antidepressant activity resides in the S-enantiomer. In addition, using a variety of in-vivo and in-vitro paradigms, it was shown that R-citalopram counteracts the effect of escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 microg/kg i.v.) blocked the suppressant effect on neuronal firing activity of both escitalopram (100 microg/kg i.v.) and paroxetine (500 microg/kg i.v.), but not fluoxetine (10 mg/kg i.v.). Interestingly, administration of R-citalopram (8 mg/kg i.p.) attenuated the increase of extracellular levels of 5-HT ([5-HT]ext) in the ventral hippocampus induced by both escitalopram (0.28 microM) and paroxetine (0.75 microM), but not fluoxetine (10 microM). In conclusion, the present in-vitro and in-vivo studies show that R-citalopram counteracts the activity of escitalopram and paroxetine, but not fluoxetine, by acting at the allosteric binding site of the 5-HT transporter, either located in the dorsal raphe nucleus or post-synaptically in the ventral hippocampus. This conclusion is strengthened by the observation that the inhibitory effect of

  18. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair.

    PubMed

    Sibanda, Bancinyane L; Chirgadze, Dimitri Y; Ascher, David B; Blundell, Tom L

    2017-02-03

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

  19. Novobiocin and Peptide Analogs of α-factor are Positive Allosteric Modulators of the Yeast G Protein-Coupled Receptor Ste2p

    PubMed Central

    Rymer, Jeffrey K.; Hauser, Melinda; Bourdon, Allen K.; Campagna, Shawn R.; Naider, Fred; Becker, Jeffrey M.

    2015-01-01

    G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR. PMID:25576192

  20. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening

    PubMed Central

    Zheng, Fudan; Robertson, Alan P.; Abongwa, Melanie; Yu, Edward W.; Martin, Richard J.

    2016-01-01

    Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization. PMID:27054065

  1. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

    PubMed

    Zheng, Fudan; Robertson, Alan P; Abongwa, Melanie; Yu, Edward W; Martin, Richard J

    2016-04-01

    Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.

  2. Baclofen and other GABAB receptor agents are allosteric modulators of the CXCL12 chemokine receptor CXCR4.

    PubMed

    Guyon, Alice; Kussrow, Amanda; Olmsted, Ian Roys; Sandoz, Guillaume; Bornhop, Darryl J; Nahon, Jean-Louis

    2013-07-10

    CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

  3. A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

    PubMed Central

    Thie, Holger

    2017-01-01

    ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

  4. Discovery and optimization of a novel series of highly CNS penetrant M4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core.

    PubMed

    Wood, Michael R; Noetzel, Meredith J; Engers, Julie L; Bollinger, Katrina A; Melancon, Bruce J; Tarr, James C; Han, Changho; West, Mary; Gregro, Alison R; Lamsal, Atin; Chang, Sichen; Ajmera, Sonia; Smith, Emery; Chase, Peter; Hodder, Peter S; Bubser, Michael; Jones, Carrie K; Hopkins, Corey R; Emmitte, Kyle A; Niswender, Colleen M; Wood, Michael W; Duggan, Mark E; Conn, P Jeffrey; Bridges, Thomas M; Lindsley, Craig W

    2016-07-01

    This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

  5. Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

    PubMed Central

    Liang, Qiansheng; Anderson, Warren D.; Jones, Shelly T.; Souza, Caio S.; Hosoume, Juliana M.; Treptow, Werner; Covarrubias, Manuel

    2015-01-01

    Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating. PMID:26599217

  6. Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.

    PubMed

    Liang, Qiansheng; Anderson, Warren D; Jones, Shelly T; Souza, Caio S; Hosoume, Juliana M; Treptow, Werner; Covarrubias, Manuel

    2015-01-01

    Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.

  7. Deconstruction of the α4β2 Nicotinic Acetylchloine (nACh) Receptor Positive Allosteric Modulator des-Formylflustrabromine (dFBr)

    PubMed Central

    German, Nadezhda; Kim, Jin-Sung; Jain, Atul; Dukat, Malgorzata; Pandya, Anshul; Ma, Yilong; Weltzin, Maegan; Schulte, Marvin K.; Glennon, Richard A.

    2011-01-01

    des -Formylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using 2-electrode voltage clamp techniques. Although the intact structure of 1 was found optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified. PMID:21905680

  8. Structure-Activity Relationship Study of Indole-2-carboxamides Identifies a Potent Allosteric Modulator for the Cannabinoid Receptor 1 (CB1)

    PubMed Central

    Mahmoud, Mariam M.; Ali, Hamed I.; Ahn, Kwang H.; Damaraju, Aparna; Samala, Sushma; Pulipati, Venkata K.; Kolluru, Srikanth; Kendall, Debra A.; Lu, Dai

    2013-01-01

    The cannabinoid CB1 receptor is involved in complex physiological functions. The discovery of CB1 allosteric modulators generates new opportunities for drug discovery targeting the pharmacologically important CB1 receptor. 5-chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569; 1) represents a new class of indole-2-carboxamides that exhibit allostery of CB1. To better understand the SAR, a group of indole-2-carboxamide analogs were synthesized and assessed for allostery of the CB1 receptor. We found that within the structure of indole-2-carboxamides, the presence of the indole ring is preferred for maintaining the modulator's high binding affinity for the allosteric site, but not for generating allostery on the orthosteric site. However, the C3 substituents of the indole-2-carboxamides significantly impact the allostery of the ligand. A robust CB1 allosteric modulator 5-chloro-N-(4-(dimethylamino)phenethyl)-3-pentyl-1H-indole-2-carboxamide (11j) was identified. It showed an equilibrium dissociation constant (KB) of 167.3 nM with a markedly high binding cooperativity factor (α=16.55) and potent antagonism of agonist-induced GTPγS binding. PMID:24053617

  9. The selective positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning and memory deficits in the Tg2576 Alzheimer's disease mouse model.

    PubMed

    Puri, Vanita; Wang, Xiaohai; Vardigan, Joshua D; Kuduk, Scott D; Uslaner, Jason M

    2015-01-01

    We have recently shown that the M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance in rodents and non-human primates administered the muscarinic receptor antagonist scopolamine. The purpose of the present experiments was to characterize the effects of PQCA in a model more relevant to the disease pathology of Alzheimer's disease. Tg2576 transgenic mice that have elevated Aβ were tested in the novel object recognition task to characterize recognition memory as a function of age and treatment with the PQCA. The effects of PQCA were compared to the acetylcholinesterase inhibitor donepezil, the standard of care for Alzheimer's disease. In addition, the effect of co-administering PQCA and donepezil was evaluated. Aged Tg2576 mice demonstrated a deficit in recognition memory that was significantly attenuated by PQCA. The positive control donepezil also reversed the deficit. Furthermore, doses of PQCA and donepezil that were inactive on their own were found to improve recognition memory when given together. These studies suggest that M1 muscarinic receptor positive allosteric modulation can ameliorate memory deficits in disease relevant models of Alzheimer's disease. These data, combined with our previous findings demonstrating PQCA improves scopolamine-induced cognitive deficits in both rodents and non-human primates, suggest that M1 positive allosteric modulators have therapeutic potential for the treatment of Alzheimer's disease.

  10. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    PubMed

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  11. Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2

    PubMed Central

    Zhuo, Ren-Gong; Peng, Peng; Liu, Xiao-Yan; Yan, Hai-Tao; Xu, Jiang-Ping; Zheng, Jian-Quan; Wei, Xiao-Li; Ma, Xiao-Yun

    2016-01-01

    In response to diverse stimuli, two-pore-domain potassium channel TREK-2 regulates cellular excitability, and hence plays a key role in mediating neuropathic pain, mood disorders and ischemia through. Although more and more input modalities are found to achieve their modulations via acting on the channel, the potential role of subunit interaction in these modulations remains to be explored. In the current study, the deletion (lack of proximal C-terminus, ΔpCt) or point mutation (G312A) was introduced into TREK-2 subunits to limit K+ conductance and used to report subunit stoichiometry. The constructs were then combined with wild type (WT) subunit to produce concatenated dimers with defined composition, and the gating kinetics of these channels to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH (pHo) were characterized. Our results show that combination of WT and ΔpCt/G312A subunits reserves similar gating properties to that of WT dimmers, suggesting that the WT subunit exerts dominant and positive effects on the mutated one, and thus the two subunits controls channel gating via a concerted cooperative manner. Further introduction of ΔpCt into the latter subunit of heterodimeric channel G312A-WT or G312A-G312A attenuated their sensitivity to 2-APB and pHo alkalization, implicating that these signals were transduced by a cis-type mechanism. Together, our findings elucidate the mechanisms for how the two subunits control the pore gating of TREK-2, in which both intersubunit concerted cooperative and cis-type manners modulate the allosteric regulations induced by 2-APB and pHo alkalization. PMID:27242438

  12. Optical filter requirements in an EML-based single-sideband PAM4 intensity-modulation and direct-detection transmission system.

    PubMed

    Chen, Hsing-Yu; Kaneda, Noriaki; Lee, Jeffrey; Chen, Jyehong; Chen, Young-Kai

    2017-03-20

    The feasibility of a single sideband (SSB) PAM4 intensity-modulation and direct-detection (IM/DD) transmission based on a CMOS ADC and DAC is experimentally demonstrated in this work. To cost effectively build a >50 Gb/s system as well as to extend the transmission distance, a low cost EML and a passive optical filter are utilized to generate the SSB signal. However, the EML-induced chirp and dispersion-induced power fading limit the requirements of the SSB filter. To separate the effect of signal-signal beating interference, filters with different roll-off factors are employed to demonstrate the performance tolerance at different transmission distance. Moreover, a high resolution spectrum analysis is proposed to depict the system limitation. Experimental results show that a minimum roll-off factor of 7 dB/10GHz is required to achieve a 51.84Gb/s 40-km transmission with only linear feed-forward equalization.

  13. Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site.

    PubMed

    Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B; Rahr, Louise M; Ahring, Philip K; Balle, Thomas; Jensen, Anders A

    2012-07-20

    The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 μM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

  14. Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors

    PubMed Central

    Ziemba, Alexis M.; Forman, Stuart A.

    2016-01-01

    Background Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range. Methods Using oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition. Results Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA. Conclusions Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites. PMID:27110714

  15. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    SciTech Connect

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  16. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains.

    PubMed

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains.

  17. Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

    SciTech Connect

    Ascenzi, Paolo Imperi, Francesco; Coletta, Massimo; Fasano, Mauro

    2008-05-02

    Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k{sub off}) is reported. In the absence of drugs, the value of k{sub off} is (1.3 {+-} 0.2) x 10{sup -4} s{sup -1}. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k{sub off} value increases to (8.6 {+-} 0.9) x 10{sup -4} s{sup -1}. From the dependence of k{sub off} on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 {+-} 0.2) x 10{sup -3} M and (6.2 {+-} 0.7) x 10{sup -5} M, respectively) were determined. The increase of k{sub off} values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.

  18. Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors

    PubMed Central

    Timmermann, DB; Sandager-Nielsen, K; Dyhring, T; Smith, M; Jacobsen, A-M; Nielsen, EØ; Grunnet, M; Christensen, JK; Peters, D; Kohlhaas, K; Olsen, GM; Ahring, PK

    2012-01-01

    BACKGROUND AND PURPOSE Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively. EXPERIMENTAL APPROACH Effects of NS9283 were evaluated in vitro using fluorescence-based Ca2+ imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats. KEY RESULTS NS9283 was shown to increase agonist-evoked response amplitude of (α4)3(β2)2 nACh receptors in electrophysiology paradigms. (α2)3(β2)2, (α2)3(β4)2 and (α4)3(β4)2 were modulated to comparable extents, but no effects were detected at α3-containing or any 2α : 3β stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHβE-sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP-disrupted pre-pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five-choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors. CONCLUSIONS AND IMPLICATIONS These results provide compelling evidence that positive allosteric modulators acting at the (α4)3(β2)2 nACh receptors can augment activity across a broad range of cognitive domains, and that α4β2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment. PMID:22506660

  19. Evaluation of peripheral versus central effects of GABAB receptor activation using a novel, positive allosteric modulator of the GABAB receptor ADX71943, a pharmacological tool compound with a fully peripheral activity profile

    PubMed Central

    Kalinichev, M; Donovan-Rodriguez, T; Girard, F; Riguet, E; Rouillier, M; Bournique, B; Haddouk, H; Mutel, V; Poli, S

    2014-01-01

    Background and Purpose The GABAB receptor agonist, baclofen, has shown promising effects in patients suffering from pain, post-traumatic stress disorder, alcoholism, overactive bladder and gastroesophageal reflux disease. However, baclofen's short duration of action and side effects limit its wider use. Here we characterized a novel, GABAB receptor positive allosteric modulator (PAM) ADX71943. Experimental Approach In vitro, ADX71943 was assessed for pharmacological activity and selectivity using recombinant and native GABAB receptors. In vivo ADX71943 was assessed in the acetic acid-induced writhing (AAW) test in mice and formalin tests (FTs) in mice and rats. Marble burying (MB) and elevated plus maze (EPM) tests, rotarod, spontaneous locomotor activity (sLMA) and body temperature (BT) tests in mice and rats were used to investigate centrally-mediated effects. Key Results In vitro, in the presence of GABA, ADX71943 increased the potency and efficacy of agonists and showed selectivity at the GABAB receptor. ADX71943 reduced pain-associated behaviours in AAW; an effect blocked by GABAB receptor antagonist CGP63360. ADX71943 reduced pain in the FT in mice and rats, but was inactive in the MB and EPM despite reaching high concentrations in plasma. ADX71943 had no effect on BT, rotarod and sLMA. Conclusions and Implications ADX71943 showed consistent and target-related efficacy in tests of disorders that have a significant peripheral component (acute and chronic pain), while having no effect in those associated with centrally-mediated anxiety-like reactivity and side effects. Thus, ADX71943 is a useful pharmacological tool for delineation of peripherally- versus centrally-mediated effects of GABAB receptor activation. PMID:24923436

  20. AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor

    PubMed Central

    Cederblad, Linda; Rosengren, Birgitta; Ryberg, Erik; Hermansson, Nils-Olov

    2015-01-01

    The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [35S]GTPγS (guanosine 5′-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of 125I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way. PMID:26656484

  1. Optimization of Novel Aza-benzimidazolone mGluR2 PAMs with Respect to LLE and PK Properties and Mitigation of CYP TDI.

    PubMed

    Pero, Joseph E; Rossi, Michael A; Kelly, Michael J; Lehman, Hannah D G F; Layton, Mark E; Garbaccio, Robert M; O'Brien, Julie A; Magliaro, Brian C; Uslaner, Jason M; Huszar, Sarah L; Fillgrove, Kerry L; Tang, Cuyue; Kuo, Yuhsin; Joyce, Leo A; Sherer, Edward C; Jacobson, Marlene A

    2016-03-10

    Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.

  2. Allosteric Modulation of protein oligomerization: an emerging approach to drug design

    NASA Astrophysics Data System (ADS)

    Gabizon, Ronen; Friedler, Assaf

    2014-03-01

    Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the "shiftides" approach developed in our lab.

  3. Potentiating mGluR5 Function with a Positive Allosteric Modulator Enhances Adaptive Learning

    ERIC Educational Resources Information Center

    Xu, Jian; Zhu, Yongling; Kraniotis, Stephen; He, Qionger; Marshall, John J.; Nomura, Toshihiro; Stauffer, Shaun R.; Lindsley, Craig W.; Conn, P. Jeffrey; Contractor, Anis

    2013-01-01

    Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity and has been associated with several neuropathological disorders. Previous work has shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction and spatial reversal learning, suggesting that mGluR5…

  4. Allosteric modulation of protein oligomerization: an emerging approach to drug design

    PubMed Central

    Gabizon, Ronen; Friedler, Assaf

    2014-01-01

    Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the “shiftides” approach developed in our lab. PMID:24790978

  5. Augmenting the spectral efficiency of enhanced PAM-DMT-based optical wireless communications.

    PubMed

    Islim, Mohamed Sufyan; Haas, Harald

    2016-05-30

    The energy efficiency of pulse-amplitude-modulated discrete multitone modulation (PAM-DMT) decreases as the modulation order of M-PAM modulation increases. Enhanced PAM-DMT (ePAM-DMT) was proposed as a solution to the reduced energy efficiency of PAM-DMT. This was achieved by allowing multiple streams of PAM-DMT to be superimposed and successively demodulated at the receiver side. In order to maintain a distortion-free unipolar ePAM-DMT system, the multiple time-domain PAM-DMT streams are required to be aligned. However, aligning the antisymmetry in ePAM-DMT is complex and results in efficiency losses. In this paper, a novel simplified method to apply the superposition modulation on M-PAM modulated discrete multitone (DMT) is introduced. Contrary to ePAM-DMT, the signal generation of the proposed system, termed augmented spectral efficiency discrete multitone (ASE-DMT), occurs in the frequency domain. This results in an improved spectral and energy efficiency. The analytical bit error rate (BER) performance bound of the proposed system is derived and compared with Monte-Carlo simulations. The system performance is shown to offer significant electrical and optical energy savings compared with ePAM-DMT and DC-biased optical orthogonal frequency division multiplexing (DCO-OFDM).

  6. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

    PubMed Central

    Pantel, Jacques; Williams, Savannah Y.; Mi, Dehui; Sebag, Julien; Corbin, Jackie D.; Weaver, C. David; Cone, Roger D.

    2011-01-01

    The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  7. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

    PubMed Central

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-01-01

    Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

  8. Allosteric Modulation of GABAA Receptors by an Anilino Enaminone in an Olfactory Center of the Mouse Brain

    PubMed Central

    Heinbockel, Thomas; Wang, Ze-Jun; Jackson-Ayotunde, Patrice L.

    2014-01-01

    In an ongoing effort to identify novel drugs that can be used as neurotherapeutic compounds, we have focused on anilino enaminones as potential anticonvulsant agents. Enaminones are organic compounds containing a conjugated system of an amine, an alkene and a ketone. Here, we review the effects of a small library of anilino enaminones on neuronal activity. Our experimental approach employs an olfactory bulb brain slice preparation using whole-cell patch-clamp recording from mitral cells in the main olfactory bulb. The main olfactory bulb is a key integrative center in the olfactory pathway. Mitral cells are the principal output neurons of the main olfactory bulb, receiving olfactory receptor neuron input at their dendrites within glomeruli, and projecting glutamatergic axons through the lateral olfactory tract to the olfactory cortex. The compounds tested are known to be effective in attenuating pentylenetetrazol (PTZ) induced convulsions in rodent models. One compound in particular, KRS-5Me-4-OCF3, evokes potent inhibition of mitral cell activity. Experiments aimed at understanding the cellular mechanism underlying the inhibitory effect revealed that KRS-5Me-4-OCF3 shifts the concentration-response curve for GABA to the left. KRS-5Me-4-OCF3 enhances GABA affinity and acts as a positive allosteric modulator of GABAA receptors. Application of a benzodiazepine site antagonist blocks the effect of KRS-5Me-4-OCF3 indicating that KRS-5Me-4-OCF3 binds at the classical benzodiazepine site to exert its pharmacological action. This anilino enaminone KRS-5Me-4-OCF3 emerges as a candidate for clinical use as an anticonvulsant agent in the battle against epileptic seizures. PMID:25525715

  9. Negative Allosteric Modulation of mGluR5 Partially Corrects Pathophysiology in a Mouse Model of Rett Syndrome.

    PubMed

    Tao, Jifang; Wu, Hao; Coronado, Amanda A; de Laittre, Elizabeth; Osterweil, Emily K; Zhang, Yi; Bear, Mark F

    2016-11-23

    Rett syndrome (RTT) is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2), an epigenetic regulator of mRNA transcription. Here, we report a test of the hypothesis of shared pathophysiology of RTT and fragile X, another monogenic cause of autism and intellectual disability. In fragile X, the loss of the mRNA translational repressor FMRP leads to exaggerated protein synthesis downstream of metabotropic glutamate receptor 5 (mGluR5). We found that mGluR5- and protein-synthesis-dependent synaptic plasticity were similarly altered in area CA1 of Mecp2 KO mice. CA1 pyramidal cell-type-specific, genome-wide profiling of ribosome-bound mRNAs was performed in wild-type and Mecp2 KO hippocampal CA1 neurons to reveal the MeCP2-regulated "translatome." We found significant overlap between ribosome-bound transcripts overexpressed in the Mecp2 KO and FMRP mRNA targets. These tended to encode long genes that were functionally related to either cytoskeleton organization or the development of neuronal connectivity. In the Fmr1 KO mouse, chronic treatment with mGluR5-negative allosteric modulators (NAMs) has been shown to ameliorate many mutant phenotypes by correcting excessive protein synthesis. In Mecp2 KO mice, we found that mGluR5 NAM treatment significantly reduced the level of overexpressed ribosome-associated transcripts, particularly those that were also FMRP targets. Some Rett phenotypes were also ameliorated by treatment, most notably hippocampal cell size and lifespan. Together, these results suggest a potential mechanistic link between MeCP2-mediated transcription regulation and mGluR5/FMRP-mediated protein translation regulation through coregulation of a subset of genes relevant to synaptic functions.

  10. The role of the second and third extracellular loops of the adenosine A1 receptor in activation and allosteric modulation.

    PubMed

    Peeters, M C; Wisse, L E; Dinaj, A; Vroling, B; Vriend, G; Ijzerman, A P

    2012-07-01

    The adenosine A1 receptor is a member of the large membrane protein family that signals through G proteins, the G protein-coupled receptors (GPCRs). GPCRs consist of seven transmembrane domains connected by three intracellular and three extracellular loops. Their N-terminus is extracellular, the C-terminal tail is in the cytoplasm. The transmembrane domains in receptor subfamilies that bind the same endogenous ligand, such as dopamine or adenosine, tend to be highly similar. In contrast, the loop regions can vary greatly, both in sequence and in length, and the role these loops have in the activation mechanism of the receptors remains unclear. Here, we investigated the activating role of the second and third extracellular loop of the human adenosine A1 receptor. By means of an (Ala)3 mutagenic scan in which consecutive sets of three amino acids were mutated into alanine residues in EL2 and a classical alanine scan in EL3, we revealed a strong regulatory role for the second extracellular loop (EL2) of the human adenosine A1 receptor. Besides many residues in the second and the third extracellular loops important for adenosine A1 receptor activation, we also identified two residues in EL2, a tryptophan and a glutamate, that affect the influence of the allosteric modulator PD81,723. These results, combined with a comparison of the different receptor loop regions, provide insight in the activation mechanism of this typical class A GPCR and further emphasize the unique pharmacological profile the loops can provide to individual receptors, even within subfamilies of GPCRs.

  11. Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice

    PubMed Central

    Pancani, Tristano; Foster, Daniel J.; Moehle, Mark S.; Bichell, Terry Jo; Bradley, Emma; Bridges, Thomas M.; Klar, Rebecca; Poslusney, Mike; Rook, Jerri M.; Daniels, J. Scott; Niswender, Colleen M.; Jones, Carrie K.; Wood, Michael R.; Bowman, Aaron B.; Lindsley, Craig W.; Xiang, Zixiu; Conn, P. Jeffrey

    2015-01-01

    Mutations that lead to Huntington’s disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M4 subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M4 positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M4 PAMs could provide a therapeutic strategy for the treatment of HD. PMID:26508634

  12. Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

    PubMed

    Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J; Panarese, Joseph D; Smith, Emery; Chase, Peter; Hodder, Peter; Niswender, Colleen; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

    2015-01-15

    Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure.

  13. Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

    PubMed

    Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

    2016-10-01

    Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium.

  14. Identification of Positive Allosteric Modulators VU0155094 (ML397) and VU0422288 (ML396) Reveals New Insights into the Biology of Metabotropic Glutamate Receptor 7

    PubMed Central

    2015-01-01

    Metabotropic glutamate receptor 7 (mGlu7) is a member of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both excitatory and inhibitory synapses, and activation of the receptor regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia. Here we report two new pan-group III mGlu positive allosteric modulators, VU0155094 and VU0422288, which show differential activity at the various group III mGlus. Additionally, both compounds show probe dependence when assessed in the presence of distinct orthosteric agonists. By pairing studies of these nonselective compounds with a synapse in the hippocampus that expresses only mGlu7, we have validated activity of these compounds in a native tissue setting. These studies provide proof-of-concept evidence that mGlu7 activity can be modulated by positive allosteric modulation, paving the way for future therapeutics development. PMID:25225882

  15. Insights into the interaction of negative allosteric modulators with the metabotropic glutamate receptor 5: discovery and computational modeling of a new series of ligands with nanomolar affinity.

    PubMed

    Anighoro, Andrew; Graziani, Davide; Bettinelli, Ilaria; Cilia, Antonio; De Toma, Carlo; Longhi, Matteo; Mangiarotti, Fabio; Menegon, Sergio; Pirona, Lorenza; Poggesi, Elena; Riva, Carlo; Rastelli, Giulio

    2015-07-01

    Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors.

  16. Allosteric Modulation of Drp1 Mechanoenzyme Assembly and Mitochondrial Fission by the Variable Domain*

    PubMed Central

    Strack, Stefan; Cribbs, J. Thomas

    2012-01-01

    The mechanoenzyme dynamin-related protein 1 (Drp1) hydrolyzes GTP to power mitochondrial fission, a process required for organelle biogenesis, quality control, transport, and apoptosis. The pleckstrin homology domain of dynamin is essential for targeting to and severing of lipid tubules, but the function of the corresponding variable domain (VD, or insert B) of Drp1 is unknown. We replaced the VD of Drp1 with a panel of linker sequences of varying length and secondary structure composition and found that the VD is dispensable for mitochondrial recruitment, association with the Drp1-anchoring protein Mff (mitochondrial fission factor), and basal and protonophore-induced mitochondrial fragmentation. Indeed, several ΔVD mutants constitutively localized to the outer mitochondrial membrane (OMM) and fragmented mitochondria more efficiently than wild-type Drp1. Consistent with an autoinhibitory role of the VD, we identified Arg-376 in the Drp1 stalk domain as necessary for Mff interaction, assembly into spirals, and mitochondrial fission. Switching the length of N- and C-terminal α-helical segments in the VD-replacing linker converted Drp1 from constitutively active and OMM-localized to inactive and cytosolic. Other hypoactive ΔVD mutants formed stable and characteristically shaped aggregates, including extended filaments. Phosphorylation of a PKA site bordering the VD disassembled the filamentous ΔVD mutant and accelerated cytosolic diffusion of full-length Drp1. We propose a model for regulation of Drp1-dependent mitochondrial fission, in which posttranslational modifications in or near the VD alter the conformation of a membrane-proximal oligomerization interface to influence Drp1 assembly rate and/or geometry. This in turn modulates Arg-376-dependent OMM targeting of Drp1 via multivalent interactions with Mff. PMID:22334657

  17. Nonclassical pharmacology of the dopamine transporter: atypical inhibitors, allosteric modulators, and partial substrates.

    PubMed

    Schmitt, Kyle C; Rothman, Richard B; Reith, Maarten E A

    2013-07-01

    The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic, and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. Because both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands possess an addictive liability equivalent to that of cocaine. However, certain recently developed ligands, such as atypical benztropine-like DAT inhibitors with reduced or even a complete lack of cocaine-like rewarding effects, suggest that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classic preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein that can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychological effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.

  18. TRR469, a potent A(1) adenosine receptor allosteric modulator, exhibits anti-nociceptive properties in acute and neuropathic pain models in mice.

    PubMed

    Vincenzi, Fabrizio; Targa, Martina; Romagnoli, Romeo; Merighi, Stefania; Gessi, Stefania; Baraldi, Pier Giovanni; Borea, Pier Andrea; Varani, Katia

    2014-06-01

    A(1) adenosine receptors (ARs) have been identified as a potential target for the development of anti-nociceptive compounds. The present study explores the analgesic effects of a novel A(1)AR positive allosteric modulator, TRR469, in different models of acute and chronic pain in mice. To evaluate the allosteric enhancement, in vitro binding experiments were performed. The anti-nociceptive properties were investigated in formalin and writhing tests, and in the streptozotocin-induced diabetic neuropathic pain model. Rotarod and catalepsy tests were used to identify potential side effects, while the functional effect of TRR469 was studied using [(3)H]-d-aspartate release from synaptosomes. TRR469 effectively inhibited nociceptive responses in the formalin and writhing tests, with effects comparable to those of the reference analgesic morphine. Isobolographic analysis of the combination of TRR469 and morphine revealed an additive interaction. TRR469 was anti-allodynic in the neuropathic pain model and did not display locomotor or cataleptic side effects. TRR469 enhanced the binding of the agonist radioligand [(3)H]-CCPA and induced a 33-fold increase of adenosine affinity in spinal cord membranes. In mouse spinal cord synaptosomes, TRR469 enhanced the inhibitory effect of A(1)AR activation on [(3)H]-d-aspartate release, a non-metabolizable analogue of glutamate. In conclusion, this research demonstrates the anti-nociceptive effect of the novel compound TRR469, one of the most potent and effective A(1)AR positive allosteric modulators so far synthesized. The use of TRR469 allows for the possibility of exploiting analgesic properties of endogenous adenosine, with a minor potential to develop the various side effects often associated with the use of direct receptor agonists.

  19. The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells

    PubMed Central

    Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

    2016-01-01

    The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation. PMID:27345502

  20. Effects of nicotine in combination with drugs described as positive allosteric nicotinic acetylcholine receptor modulators in vitro: discriminative stimulus and hypothermic effects in mice.

    PubMed

    Moerke, Megan J; de Moura, Fernando B; Koek, Wouter; McMahon, Lance R

    2016-09-05

    Some drugs that are positive allosteric nAChR modulators in vitro, desformylflustrabromine (dFBr), PNU-120596 and LY 2087101, have not been fully characterized in vivo. These drugs were examined for their capacity to share or modify the hypothermic and discriminative stimulus effects of nicotine (1mg/kg s.c.) in male C57Bl/6J mice. Nicotine, dFBr, and PNU-120596 produced significant hypothermia, whereas LY 2087101 (up to 100mg/kg) did not. Nicotine dose-dependently increased nicotine-appropriate responding and decreased response rate; the respective ED50 values were 0.56mg/kg and 0.91mg/kg. The modulators produced no more than 38% nicotine-appropriate responding up to doses that disrupted operant responding. Rank order potency was the same for hypothermia and rate-decreasing effects: nicotine>dFBr>PNU-120596=LY 2087101. Mecamylamine and the α4β2 nAChR antagonist dihydro-β-erythroidine, but not the α7 antagonist methyllycaconitine, antagonized the hypothermic effects of nicotine. In contrast, mecamylamine did not antagonize the hypothermic effects of the modulators. The combined discriminative stimulus effects of DFBr and nicotine were synergistic, whereas the combined hypothermic effects of nicotine with either dFBr or PNU-120596 were infra-additive. PNU-120596 did not modify the nicotine discriminative stimulus, and LY 2087101 did not significantly modify either effect of nicotine. Positive modulation of nicotine at nAChRs by PNU-120596 and LY 2087101 in vitro does not appear to confer enhancement of the nAChR-mediated hypothermic or discriminative stimulus effects of nicotine. However, dFBr appears to be a positive allosteric modulator of some behavioral effects of nicotine at doses of dFBr smaller than the doses producing unwanted effects (e.g. hypothermia) through non-nAChR mechanisms.

  1. Alpha7 nicotinic acetylcholine receptor agonists and PAMs as adjunctive treatment in schizophrenia. An experimental study.

    PubMed

    Marcus, Monica M; Björkholm, Carl; Malmerfelt, Anna; Möller, Annie; Påhlsson, Ninni; Konradsson-Geuken, Åsa; Feltmann, Kristin; Jardemark, Kent; Schilström, Björn; Svensson, Torgny H

    2016-09-01

    Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Therefore, there has been a large interest in developing drugs affecting the α7 nAChRs for schizophrenia. In the present study we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive allosteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. Moreover we also investigated their utility as adjunct treatment in depression in combination with the SSRI citalopram. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release. Finally, PNU282987 reduced immobility in the forced swim test, indicating an antidepressant-like effect. Taken together, our data support the utility of drugs targeting the α7 nAChRs, perhaps especially α7 PAMs, to potentiate the effect of atypical antipsychotic drugs. Moreover, our data suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia and depression.

  2. 3.375-Gb/s RGB-LED based WDM visible light communication system employing PAM-8 modulation with phase shifted Manchester coding.

    PubMed

    Chi, Nan; Zhang, Mengjie; Zhou, Yingjun; Zhao, Jiaqi

    2016-09-19

    Optical background noise and second-order nonlinear distortions are two main challenges faced by indoor high-speed VLC system. In this paper, a novel phase shifted Manchester (PS-Manchester) coding based on PAM-8 is proposed and experimentally demonstrated to mitigate these noise and distortions. With the aid of PS-Manchester coding and WDM, a total data rate of 3.375-Gb/s can be successfully achieved in the RGB-LED based VLC system. The BER is under 7% HD-FEC limit of 3.8x10-3 after 1-m indoor free space transmission. To the best of our knowledge, this is the highest data rate ever achieved in PAM VLC systems.

  3. On the g-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation.

    PubMed

    Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Rivera, Alicia; Van Craenenbroeck, Kathleen; Tarakanov, Alexander O; Agnati, Luigi F; Fuxe, Kjell

    2013-01-01

    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β -arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

  4. On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

    PubMed Central

    Borroto-Escuela, Dasiel O.; Romero-Fernandez, Wilber; Rivera, Alicia; Van Craenenbroeck, Kathleen; Tarakanov, Alexander O.; Agnati, Luigi F.; Fuxe, Kjell

    2013-01-01

    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia. PMID:23956775

  5. Diarylureas as Allosteric Modulators of the Cannabinoid CB1 Receptor: Structure–Activity Relationship Studies on 1-(4-Chlorophenyl)-3-{3-[6-(pyrrolidin-1-yl)pyridin-2-yl]phenyl}urea (PSNCBAM-1)

    PubMed Central

    2015-01-01

    The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [3H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor. PMID:25162172

  6. The Effect of Thymoquinone, α7 Receptor Agonist and α7 Receptor Allosteric Modulator on the Cerebral Cortex in Experimentally Induced Alzheimer’s Disease in Relation to MSCs Activation

    PubMed Central

    AbdEl Fattah, Lamiaa Ibrahim; Zickri, Maha Baligh; Aal, Lobna Abdel; Heikal, Ola; Osama, Esraa

    2016-01-01

    Background and Objectives Alzheimer’s disease (AD) is the most common form of dementia among older persons. Thymoquinone (TQ) has anti-inflammatory, anticonvulsant and antioxidant activity. A novel α7 nicotinic acetyl choline receptor (α7 nAChR ) agonist (PNU- 282987) have been identified to enhance the cognitive performance. An alternative treatment strategy via compounds known as nicotinic “positive allosteric modulators” (PAMs) has been reported. This study was designed to investigate the combination of PAM of α7 nAChRs with PNU- 282987 or with TQ as a possible treatment for AD in rat. Methods 48 male albino rats were divided into 4 groups. Group I (Control), Group II received lipopolysaccharide, 0.8 mg/kg by intraperitoneal injection (IPI) once, Group III received TQ 10 mg/kg by IPI, Group IV received PNU-120596 1 mg/kg by IPI, in addition to PNU-282987 1 mg/kg by IPI in subgroup IVa and TQ in subgroup b. All treatment drugs were given for 5 days. Results Acidophilic masses, deformed neurons, Congo red +ve masses and reduced Phospho-CREB immunoexpression were seen in group II. All changes regressed by treatment. Some CD44 +ve cells were noticed in group II and few +ve cells in subgroup IVa, that became multiple in group III and subgroup IVb. The histological, histochemical and immunohistochemical changes were confirmed statistically and significant differences were recorded. Conclusions TQ or α7 nAChR agonist combined with PAM can have an important role in treatment of AD that is superior to thymoquinone alone. Exceptionally, TQ single or combined with PAM proved activation of MSC. PMID:27572711

  7. The Role of mGlu Receptors in Hippocampal Plasticity Deficits in Neurological and Psychiatric Disorders: Implications for Allosteric Modulators as Novel Therapeutic Strategies

    PubMed Central

    Senter, Rebecca K.; Ghoshal, Ayan; Walker, Adam G.; Xiang, Zixiu; Niswender, Colleen M.; Conn, P. Jeffrey

    2016-01-01

    Long-term potentiation (LTP) and long-term depression (LTD) are two distinct forms of synaptic plasticity that have been extensively characterized at the Schaffer collateral-CA1 (SC-CA1) synapse and the mossy fiber (MF)-CA3 synapse within the hippocampus, and are postulated to be the molecular underpinning for several cognitive functions. Deficits in LTP and LTD have been implicated in the pathophysiology of several neurological and psychiatric disorders. Therefore, there has been a large effort focused on developing an understanding of the mechanisms underlying these forms of plasticity and novel therapeutic strategies that improve or rescue these plasticity deficits. Among many other targets, the metabotropic glutamate (mGlu) receptors show promise as novel therapeutic candidates for the treatment of these disorders. Among the eight distinct mGlu receptor subtypes (mGlu1-8), the mGlu1,2,3,5,7 subtypes are expressed throughout the hippocampus and have been shown to play important roles in the regulation of synaptic plasticity in this brain area. However, development of therapeutic agents that target these mGlu receptors has been hampered by a lack of subtype-selective compounds. Recently, discovery of allosteric modulators of mGlu receptors has provided novel ligands that are highly selective for individual mGlu receptor subtypes. The mGlu receptors modulate the multiple forms of synaptic plasticity at both SC-CA1 and MF synapses and allosteric modulators of mGlu receptors have emerged as potential therapeutic agents that may rescue plasticity deficits and improve cognitive function in patients suffering from multiple neurological and psychiatric disorders. PMID:27296640

  8. DRUG FOCUS: S 18986: A positive allosteric modulator of AMPA-type glutamate receptors pharmacological profile of a novel cognitive enhancer.

    PubMed

    Bernard, Katy; Danober, Laurence; Thomas, Jean-Yves; Lebrun, Cécile; Muñoz, Carmen; Cordi, Alex; Desos, Patrice; Lestage, Pierre; Morain, Philippe

    2010-10-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) type glutamate receptors are critical for synaptic plasticity and induction of long-term potentiation (LTP), considered as one of the synaptic mechanisms underlying learning and memory. Positive allosteric modulators of AMPA receptors could provide a therapeutic approach to the treatment of cognitive disorders resulting from aging and/or neurodegenerative diseases, such as Alzheimer disease (AD). Several AMPA potentiators have been described in the last decade, but for the moment their clinical efficacy has not been demonstrated due to the complexity of the target, AMPA receptors, and the difficulty in studying cognition in animals and humans. A better understanding of the mechanism of action of this type of drug remains an important issue, if knowledge of these compounds is to be increased and if this novel therapeutic approach is to be an interesting research area. Among the AMPA potentiators, S 18986 is emerging as a new selective positive allosteric modulator of AMPA-type glutamate receptors. S 18986, as with other positive AMPA receptor modulators, increased induction and maintenance of LTP in the hippocampus as well as the expression of brain-derived neurotrophic factor (BDNF) both in vitro and in vivo. Its cognitive-enhancing properties have been demonstrated in various behavioral models (procedural, spatial, "episodic," working, and relational/declarative memory) in young-adult and aged rodents. It is interesting to note that memory-enhancing effects appeared more robust in middle-aged animals compared with aged ones and in "episodic" and spatial memory tasks. From these results, S 18986 is expected to treat memory deficits associated with early cerebral aging and neurological diseases in elderly people.

  9. The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins

    PubMed Central

    Merret, Rémy; Martino, Luigi; Bousquet-Antonelli, Cécile; Fneich, Sara; Descombin, Julie; Billey, Élodie; Conte, Maria R.; Deragon, Jean-Marc

    2013-01-01

    La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3′ UTRs and led to their neofunctionalization. PMID:23148093

  10. The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins.

    PubMed

    Merret, Rémy; Martino, Luigi; Bousquet-Antonelli, Cécile; Fneich, Sara; Descombin, Julie; Billey, Elodie; Conte, Maria R; Deragon, Jean-Marc

    2013-01-01

    La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3' UTRs and led to their neofunctionalization.

  11. Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-D-aspartate receptor (NMDAR) containing GluN2B subunit.

    PubMed

    Buemi, Maria Rosa; De Luca, Laura; Ferro, Stefania; Russo, Emilio; De Sarro, Giovambattista; Gitto, Rosaria

    2016-04-01

    Negative allosteric modulators (NAMs) of GluN2B-containing NMDARs provide pharmacological tools for the treatment of chronic neurodegenerative diseases. Novel NAMs have been designed on the basis of computational studies focused on the 'hit compound' 3. This series of indoles has been tested in competition assay. Compounds 16 and 17 were the most active ligands (IC50 values of 83 nM and 71 nM, respectively) and they showed a potency close to that of reference compounds ifenprodil (1, IC50=47 nM) and 3 (IC50=25 nM). Furthermore, docking studies have been performed for active ligand 16 and the results were in a good agreement with biological data.

  12. The GABA(B) receptor agonist, baclofen, and the positive allosteric modulator, CGP7930, inhibit visceral pain-related responses to colorectal distension in rats.

    PubMed

    Brusberg, Mikael; Ravnefjord, Anna; Martinsson, Rakel; Larsson, Håkan; Martinez, Vicente; Lindström, Erik

    2009-02-01

    Activation of GABA(B) receptors by the selective agonist baclofen produces anti-nociceptive effects in animal models of somatic pain. The aim of the present study was to evaluate the effect of baclofen and the GABA(B) receptor positive allosteric modulator CGP7930 on pseudo-affective responses to colorectal distension in rats. Female Sprague-Dawley rats were subjected to repeated, noxious colorectal distension (CRD) (12 distensions at 80 mmHg, for 30 s with 5 min intervals). The visceromotor response (VMR) and cardiovascular responses (mean arterial blood pressure (ABP) and heart rate (HR)) to CRD were monitored in conscious, telemetrized animals. Baclofen (0.3-3 micromol/kg, i.v.) reduced the VMR to CRD dose-dependently, reaching a 61% maximal inhibition (p < 0.001). The highest doses of baclofen attenuated CRD-evoked increases in ABP by 17% (p > 0.05) and reduced the change in HR by 48% (p < 0.01). CGP7930 (3-30 micromol/kg, i.v.) reduced the VMR to CRD in a dose-dependent fashion with a maximal inhibition of 31% (p < 0.05). The highest dose of CGP7930 also attenuated the increase in ABP by 18% (p > 0.05) and inhibited the increase in HR by 24% (p < 0.05) associated with CRD. Neither baclofen nor CGP7930 affected colorectal compliance. The results suggest that activation of GABA(B) receptors produces anti-nociceptive effects in a rat model of mechanically induced visceral pain. While CGP7930 was less efficacious than baclofen overall, positive allosteric modulation of GABA(B) receptors may represent a valid approach in the treatment of visceral pain conditions, with the possibility of an improved safety profile compared to full agonism.

  13. Behavioral Effects of the Benzodiazepine-Positive Allosteric Modulator SH-053-2’F-S-CH3 in an Immune-Mediated Neurodevelopmental Disruption Model

    PubMed Central

    Richetto, Juliet; Labouesse, Marie A.; Poe, Michael M.; Cook, James M.; Grace, Anthony A.; Riva, Marco A.

    2015-01-01

    Background: Impaired γ-aminobutyric acid (GABA) signaling may contribute to the emergence of cognitive deficits and subcortical dopaminergic hyperactivity in patients with schizophrenia and related psychotic disorders. Against this background, it has been proposed that pharmacological interventions targeting GABAergic dysfunctions may prove useful in correcting such cognitive impairments and dopaminergic imbalances. Methods: Here, we explored possible beneficial effects of the benzodiazepine-positive allosteric modulator SH-053-2’F-S-CH3, with partial selectivity at the α2, α3, and α5 subunits of the GABAA receptor in an immune-mediated neurodevelopmental disruption model. The model is based on prenatal administration of the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)] in mice, which is known to capture various GABAergic, dopamine-related, and cognitive abnormalities implicated in schizophrenia and related disorders. Results: Real-time polymerase chain reaction analyses confirmed the expected alterations in GABAA receptor α subunit gene expression in the medial prefrontal cortices and ventral hippocampi of adult poly(I:C) offspring relative to control offspring. Systemic administration of SH-053-2’F-S-CH3 failed to normalize the poly(I:C)-induced deficits in working memory and social interaction, but instead impaired performance in these cognitive and behavioral domains both in control and poly(I:C) offspring. In contrast, SH-053-2’F-S-CH3 was highly effective in mitigating the poly(I:C)-induced amphetamine hypersensitivity phenotype without causing side effects in control offspring. Conclusions: Our preclinical data suggest that benzodiazepine-like positive allosteric modulators with activity at the α2, α3, and α5 subunits of the GABAA receptor may be particularly useful in correcting pathological overactivity of the dopaminergic system, but they may be ineffective in targeting multiple pathological domains that involve the co

  14. Allosteric Modulation of Related Ligand-Gated Ion Channels Synergistically Induces Long-Term Potentiation in the Hippocampus and Enhances Cognition

    PubMed Central

    Gu, Zhenglin; Yoshimura, Ryan F.; Villegier, Anne-Sophie; Hogenkamp, Derk J.; Whittemore, Edward R.; Huang, Jin-Cheng; Tran, Minhtam B.; Belluzzi, James D.; Yakel, Jerrel L.; Gee, Kelvin W.

    2011-01-01

    α5 Subunit-containing GABAA receptors (GABAARs) and α7 neuronal nicotinic-acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels (LGICs) that mediate cognitive and attentional processes in the hippocampus. α5 GABAARs alter network activity by tonic inhibition of CA1/CA3 pyramidal cells of the hippocampus. Postsynaptic α7 nAChRs in the hippocampus regulate inhibitory GABAergic interneuron activity required for synchronization of pyramidal neurons in the CA1, whereas presynaptic α7 nAChRs regulate glutamate release. Can simultaneous allosteric modulation of these LGICs produce synergistic effects on cognition? We show that combined transient application of two allosteric modulators that individually 1) inhibit α5 GABAARs and 2) enhance α7 nAChRs causes long-term potentiation (LTP) of mossy fiber stimulation-induced excitatory postsynaptic currents (EPSC) from CA1 pyramidal neurons of rat hippocampal slices. The LTP effect evoked by two compounds is replicated by 3-(2,5-difluorophenyl)-6-(N-ethylindol-5-yl)-1,2,4-triazolo[4,3-b]pyridazine (522-054), a compound we designed to simultaneously inhibit α5 GABAARs and enhance α7 nAChRs. Selective antagonists for either receptor block sustained EPSC potentiation produced by 522-054. In vivo, 522-054 enhances performance in the radial arm maze and facilitates attentional states in the five-choice serial reaction time trial with similar receptor antagonist sensitivity. These observations may translate into therapeutic utility of dual action compounds in diseases of hippocampal-based cognitive impairment. PMID:21159751

  15. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.

    PubMed

    Artsimovitch, Irina; Vassylyeva, Marina N; Svetlov, Dmitri; Svetlov, Vladimir; Perederina, Anna; Igarashi, Noriyuki; Matsugaki, Naohiro; Wakatsuki, Soichi; Tahirov, Tahir H; Vassylyev, Dmitry G

    2005-08-12

    Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.

  16. Allosteric modulation of the effects of the 5-HT reuptake inhibitor escitalopram on the rat hippocampal synaptic plasticity.

    PubMed

    Mnie-Filali, Ouissame; El Mansari, Mostafa; Espana, Agnès; Sànchez, Connie; Haddjeri, Nasser

    2006-02-27

    The present in vivo electrophysiological studies in anesthetized rat were undertaken to assess the effects of the selective serotonin (5-HT) reuptake inhibitor (SSRI) escitalopram alone or in combination with the R-citalopram (the S- and R-enantiomers of citalopram), on both long-term potentiation (LTP) in the CA(1) region of dorsal hippocampus and spontaneous firing activity of dorsal raphe (DR) 5-HT neurons. At the postsynaptic level, neither escitalopram (10 mg/kg, i.p.) nor R-citalopram (20 mg/kg, i.p.) modified basal synaptic transmission but only escitalopram impaired LTP expression. Importantly, R-citalopram counteracted significantly the escitalopram-induced decrease of LTP. At the pre-synaptic level, escitalopram (25-75 microg/kg, i.v.) dose-dependently suppressed the spontaneous firing activity of DR 5-HT neurons and this suppressant effect was significantly prevented by a prior injection of R-citalopram (10 mg/kg, i.p.). These results support a role of allosteric binding sites of 5-HT transporter in the regulation of long-lasting CA(1) synaptic plasticity and DR 5-HT neuronal firing activity.

  17. Allosteric Dynamic Control of Binding

    PubMed Central

    Sumbul, Fidan; Acuner-Ozbabacan, Saliha Ece; Haliloglu, Turkan

    2015-01-01

    Proteins have a highly dynamic nature and there is a complex interrelation between their structural dynamics and binding behavior. By assuming various conformational ensembles, they perform both local and global fluctuations to interact with other proteins in a dynamic infrastructure adapted to functional motion. Here, we show that there is a significant association between allosteric mutations, which lead to high-binding-affinity changes, and the hinge positions of global modes, as revealed by a large-scale statistical analysis of data in the Structural Kinetic and Energetic Database of Mutant Protein Interactions (SKEMPI). We further examined the mechanism of allosteric dynamics by conducting studies on human growth hormone (hGH) and pyrin domain (PYD), and the results show how mutations at the hinge regions could allosterically affect the binding-site dynamics or induce alternative binding modes by modifying the ensemble of accessible conformations. The long-range dissemination of perturbations in local chemistry or physical interactions through an impact on global dynamics can restore the allosteric dynamics. Our findings suggest a mechanism for the coupling of structural dynamics to the modulation of protein interactions, which remains a critical phenomenon in understanding the effect of mutations that lead to functional changes in proteins. PMID:26338442

  18. PAM sequence design for dimmable visible light communication

    NASA Astrophysics Data System (ADS)

    Nuo, Huang; Wang, Jun-Bo; Wang, Jin-Yuan; Guan, Rui; Chen, Ming

    2017-02-01

    In current visible light communication (VLC) systems employing intensity modulation and direct detection (IM/DD), the transmitted optical intensity signal must satisfy the nonnegativity, peak optical intensity and illumination constraints. By taking into account the three constraints, we first present the signal space for the pulse amplitude modulated (PAM) sequences in the maximum flickering time period (MFTP). Then, to minimize the error probability of the VLC systems, we seek to find the PAM sequences providing the largest minimum Euclidean distance. Since the objective function is nonconvex and nondifferentiable, it is difficult to solve the original optimization problem directly. Thus, two methods corresponding to the joint design and greedy algorithm, are proposed to design the PAM sequences in VLC. The two methods offer a tradeoff between the symbol error rate (SER) performance and design complexity.

  19. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease

    PubMed Central

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD. PMID:27293955

  20. Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

    PubMed

    Palanisamy, Gopinath S; Marcek, John M; Cappon, Gregg D; Whritenour, Jessica; Shaffer, Christopher L; Brady, Joseph T; Houle, Christopher

    2015-10-01

    Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

  1. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease.

    PubMed

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

  2. Separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ9-THC in humans discriminating Δ9-THC

    PubMed Central

    Lile, Joshua A.; Kelly, Thomas H.; Hays, Lon R.

    2014-01-01

    Background Our previous research suggested the involvement γ-aminobutyric acid (GABA), in particular the GABAB receptor subtype, in the interoceptive effects of Δ9-tetrahydrocannabinol (Δ9-THC). The aim of the present study was to determine the potential involvement of the GABAA receptor subtype by assessing the separate and combined effects of the GABAA positive allosteric modulator diazepam and Δ9-THC using pharmacologically selective drug-discrimination procedures. Methods Ten cannabis users learned to discriminate 30 mg oral Δ9-THC from placebo and then received diazepam (5 and 10 mg), Δ9-THC (5, 15 and 30 mg) and placebo, alone and in combination. Self-report, task performance and physiological measures were also collected. Results Δ9-THC functioned as a discriminative stimulus, produced subjective effects typically associated with cannabinoids (e.g., High, Stoned, Like Drug) and elevated heart rate. Diazepam alone impaired performance on psychomotor performance tasks and increased ratings on a limited number of self-report questionnaire items (e.g., Any Effect, Sedated), but did not substitute for the Δ9-THC discriminative stimulus or alter the Δ9-THC discrimination dose-response function. Similarly, diazepam had limited impact on the other behavioral effects of Δ9-THC. Conclusions These results suggest that the GABAA receptor subtype has minimal involvement in the interoceptive effects of Δ9-THC, and by extension cannabis, in humans. PMID:25124305

  3. Pharmacokinetic and pharmacodynamic characterisation of JNJ-40411813, a positive allosteric modulator of mGluR2, in two randomised, double-blind phase-I studies.

    PubMed

    Salih, Hiba; Anghelescu, Ion; Kezic, Iva; Sinha, Vikash; Hoeben, Eef; Van Nueten, Luc; De Smedt, Heidi; De Boer, Peter

    2015-04-01

    Metabotropic glutamate receptor-2 positive allosteric modulator, JNJ-40411813 (ADX71149), was characterised for clinical effects in healthy volunteers in two phase-1 studies. In study 1, healthy men received 50-, 100-, 150- or 225 mg and women received 100 mg JNJ-40411813 (n=6, each cohort) or placebo (n=2, each cohort) twice daily for seven days; smoking men (n=30) received placebo twice daily on days 1-7, 100 mg JNJ-40411813 (n=20) or placebo (n=10) on days 8-14. In study 2, healthy men received intravenous 0.005 mg/kg S(+) ketamine over 60 min at 3 (n=24; cohort 1), 12 h (n=8; cohort 3), and 24 h (n=8; cohort 2) after a single oral dose of 500 mg JNJ-40411813 or placebo. The pharmacokinetics and effects of JNJ-40411813 on cognition and subjective awareness were evaluated. Plasma JNJ-40411813 exposure was dose-dependent, t max ranged from 3-4 h and t 1/2 19.4-34.2 h across the dose levels. JNJ-40411813 significantly (p=0.02) reduced continuity of attention score (150 mg dose) and ameliorated smoking withdrawal-induced changes in power of attention and quality of episodic memory versus placebo. A modest reduction in alertness was observed at 150-225 mg doses, JNJ-40411813 (500 mg) reduced S(+) ketamine-induced negative symptoms by approximately 43% and 30% in cohorts 1 and 3, respectively. JNJ-40411813 was generally well-tolerated.

  4. Selective Remediation of Reversal Learning Deficits in the Neurodevelopmental MAM Model of Schizophrenia by a Novel mGlu5 Positive Allosteric Modulator

    PubMed Central

    Gastambide, Francois; Cotel, Marie-Caroline; Gilmour, Gary; O'Neill, Michael J; Robbins, Trevor W; Tricklebank, Mark D

    2012-01-01

    Based on the glutamatergic hypothesis of schizophrenia we assessed the effects of a novel mGlu5 positive allosteric modulator, LSN2463359 [N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide] on deficits in cognitive flexibility in two distinct rodent models of schizophrenia, the neurodevelopmental MAM E17 model and the acute PCP model. Cognitive flexibility was measured with the intra-dimensional and extra-dimensional set-shifting and reversal learning digging paradigm. Regional effects of MAM on the expression of parvalbumin-positive cells (PV) and mGlu5 receptors were also examined, to further characterize the model. Results showed that LSN2463359 selectively attenuated reversal learning deficits in the MAM but not acute PCP model. Whilst both models led to deficits in reversal learning and extra-dimensional set-shifting, the reversal impairments were qualitatively distinct, with MAM increasing perseverative responding, whereas the PCP deficit was mainly due to the inability of rats to maintain reinforced choice behavior. Reduction of PV and mGlu5 expression was found in the MAM model in several regions of importance in schizophrenia, such as the orbitofrontal and medial prefrontal cortex, which also mediate reversal learning and extra-dimensional set-shifting. The present findings confirm that the positive modulation of mGlu5 receptors may have beneficial effects in the treatment of certain aspects of cognitive impairment associated with schizophrenia. This study also illustrates the importance of studying putative cognitive enhancing drug effects in a number of models which may have implications for the future development of the compound. PMID:22129780

  5. Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

    PubMed Central

    Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

    2013-01-01

    Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

  6. Selective remediation of reversal learning deficits in the neurodevelopmental MAM model of schizophrenia by a novel mGlu5 positive allosteric modulator.

    PubMed

    Gastambide, Francois; Cotel, Marie-Caroline; Gilmour, Gary; O'Neill, Michael J; Robbins, Trevor W; Tricklebank, Mark D

    2012-03-01

    Based on the glutamatergic hypothesis of schizophrenia we assessed the effects of a novel mGlu5 positive allosteric modulator, LSN2463359 [N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide] on deficits in cognitive flexibility in two distinct rodent models of schizophrenia, the neurodevelopmental MAM E17 model and the acute PCP model. Cognitive flexibility was measured with the intra-dimensional and extra-dimensional set-shifting and reversal learning digging paradigm. Regional effects of MAM on the expression of parvalbumin-positive cells (PV) and mGlu5 receptors were also examined, to further characterize the model. Results showed that LSN2463359 selectively attenuated reversal learning deficits in the MAM but not acute PCP model. Whilst both models led to deficits in reversal learning and extra-dimensional set-shifting, the reversal impairments were qualitatively distinct, with MAM increasing perseverative responding, whereas the PCP deficit was mainly due to the inability of rats to maintain reinforced choice behavior. Reduction of PV and mGlu5 expression was found in the MAM model in several regions of importance in schizophrenia, such as the orbitofrontal and medial prefrontal cortex, which also mediate reversal learning and extra-dimensional set-shifting. The present findings confirm that the positive modulation of mGlu5 receptors may have beneficial effects in the treatment of certain aspects of cognitive impairment associated with schizophrenia. This study also illustrates the importance of studying putative cognitive enhancing drug effects in a number of models which may have implications for the future development of the compound.

  7. Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism.

    PubMed

    Silverman, Jill L; Smith, Daniel G; Rizzo, Stacey J Sukoff; Karras, Michael N; Turner, Sarah M; Tolu, Seda S; Bryce, Dianne K; Smith, Deborah L; Fonseca, Kari; Ring, Robert H; Crawley, Jacqueline N

    2012-04-25

    Neurodevelopmental disorders such as autism and fragile X syndrome were long thought to be medically untreatable, on the assumption that brain dysfunctions were immutably hardwired before diagnosis. Recent revelations that many cases of autism are caused by mutations in genes that control the ongoing formation and maturation of synapses have challenged this dogma. Antagonists of metabotropic glutamate receptor subtype 5 (mGluR5), which modulate excitatory neurotransmission, are in clinical trials for fragile X syndrome, a major genetic cause of intellectual disabilities. About 30% of patients with fragile X syndrome meet the diagnostic criteria for autism. Reasoning by analogy, we considered the mGluR5 receptor as a potential target for intervention in autism. We used BTBR T+tf/J (BTBR) mice, an established model with robust behavioral phenotypes relevant to the three diagnostic behavioral symptoms of autism--unusual social interactions, impaired communication, and repetitive behaviors--to probe the efficacy of a selective negative allosteric modulator of the mGluR5 receptor, GRN-529. GRN-529 reduced repetitive behaviors in three cohorts of BTBR mice at doses that did not induce sedation in control assays of open field locomotion. In addition, the same nonsedating doses reduced the spontaneous stereotyped jumping that characterizes a second inbred strain of mice, C58/J. Further, GRN-529 partially reversed the striking lack of sociability in BTBR mice on some parameters of social approach and reciprocal social interactions. These findings raise the possibility that a single targeted pharmacological intervention may alleviate multiple diagnostic behavioral symptoms of autism.

  8. Allosteric modulation of [3H]-CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain

    PubMed Central

    Mugnaini, Manolo; Meoni, Paolo; Bunnemann, Bernd; Corsi, Mauro; Bowery, Norman G

    2001-01-01

    Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques.In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [3H]-CGP39653 binding, but competitively reversed the high affinity component of [3H]-CGP39653 binding inhibition by glycine, with a pKB value of 8.38, in line with its affinity for the glycine site (pKi=8.49 vs [3H]-glycine). Glycine (10 μM) significantly decreased [3H]-CGP39653 affinity for the NMDA receptor (with no change in the Bmax), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test).In rat brain sections the addition of GV150526A (30 μM) to the incubation medium increased [3H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [3H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation.Whole brain autoradiography revealed an uneven distribution of [3H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [3H]-CGP39653 binding was increased to 216% of

  9. A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators*

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Kessler, Markus; Stone, Leslie M.; Arai, Amy C.; Partin, Kathryn M.

    2014-01-01

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators. PMID:24550387

  10. A charge-inverting mutation in the "linker" region of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators.

    PubMed

    Harms, Jonathan E; Benveniste, Morris; Kessler, Markus; Stone, Leslie M; Arai, Amy C; Partin, Kathryn M

    2014-04-11

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.

  11. The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents

    PubMed Central

    Rigo, J-M; Hans, G; Nguyen, L; Rocher, V; Belachew, S; Malgrange, B; Leprince, P; Moonen, G; Selak, I; Matagne, A; Klitgaard, H

    2002-01-01

    In this study in vitro and in vivo approaches were combined in order to investigate if the anti-epileptic mechanism(s) of action of levetiracetam (LEV; Keppra®) may involve modulation of inhibitory neurotransmission.GABA- and glycine-gated currents were studied in vitro using whole-cell patch-clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound-susceptible mice. The effect of LEV was compared with reference anti-epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide.LEV contrasted the reference AEDs by an absence of any direct effect on glycine-gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA-gated currents. A similar action on GABA-elicited currents was observed with the reference AEDs, except ethosuximide.These minor direct effects contrasted with a potent ability of LEV (EC50=1 – 10 μM) to reverse the inhibitory effects of the negative allosteric modulators zinc and β-carbolines on both GABAA and glycine receptor-mediated responses.Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of β-carbolines on GABA-gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine-gated currents and only clonazepam and phenobarbital inhibited the action of DMCM.LEV (17 mg kg−1) produced a potent suppression of sound-induced clonic convulsions in mice. This protective effect was significantly abolished by co-administration of the β-carboline FG 7142, from a dose of 5 mg kg−1. In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg−1) was without any effect on the protection

  12. Allosteric modulation of the activity of the glucagon-like peptide-1 (GLP-1) metabolite GLP-1 9-36 amide at the GLP-1 receptor.

    PubMed

    Li, Naichang; Lu, Jing; Willars, Gary B

    2012-01-01

    Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrients has many physiological effects but particularly enhances glucose-dependent insulin release through the GLP-1 receptor (GLP-1R). GLP-1 7-36 amide, the predominant circulating active form of GLP-1, is rapidly truncated by dipeptidyl peptidase-4 to GLP-1 9-36 amide, which is generally considered inactive. Given its physiological roles, the GLP-1R is targeted for treatment of type 2 diabetes. Recently 'compound 2' has been described as both an agonist and positive allosteric modulator of GLP-1 7-36 amide affinity, but not potency, at the GLP-1R. Importantly, we demonstrated previously that exendin 9-39, generally considered a GLP-1R antagonist, enhances compound 2 efficacy (or vice versa) at the GLP-1R. Given that GLP-1 9-36 amide is the major circulating form of GLP-1 post-prandially and is a low affinity weak partial agonist or antagonist at the GLP-1R, we investigated interaction between this metabolite and compound 2 in a cell line with recombinant expression of the human GLP-1R and the rat insulinoma cell line, INS-1E, with native expression of the GLP-1R. We show compound 2 markedly enhances efficacy and potency of GLP-1 9-36 amide for key cellular responses including AMP generation, Ca(2+) signaling and extracellular signal-regulated kinase. Thus, metabolites of peptide hormones including GLP-1 that are often considered inactive may provide a means of manipulating key aspects of receptor function and a novel therapeutic strategy.

  13. The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development.

    PubMed

    Keavy, Deborah; Bristow, Linda J; Sivarao, Digavalli V; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Weed, Michael R

    2016-01-01

    The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans.

  14. The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor.

    PubMed

    Hempel, Christoph; Nörenberg, Wolfgang; Sobottka, Helga; Urban, Nicole; Nicke, Annette; Fischer, Wolfgang; Schaefer, Michael

    2013-12-01

    P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

  15. The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development

    PubMed Central

    Keavy, Deborah; Bristow, Linda J.; Sivarao, Digavalli V.; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E.; Weed, Michael R.

    2016-01-01

    The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans. PMID:27035340

  16. Lu AF21934, a positive allosteric modulator of mGlu4 receptors, reduces the harmaline-induced hyperactivity but not tremor in rats.

    PubMed

    Ossowska, Krystyna; Wardas, Jadwiga; Berghauzen-Maciejewska, Klemencja; Głowacka, Urszula; Kuter, Katarzyna; Pilc, Andrzej; Zorn, Stevin H; Doller, Dario

    2014-08-01

    Harmaline induces tremor in animals resembling essential tremor which has been suggested to result from activation of the glutamatergic olivo-cerebellar projection. The aim of the present study was to examine the effects of systemic administration of Lu AF21934, a brain-penetrating positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu4), on the harmaline-induced tremor and other forms of motor activity in rats using fully automated Force Plate Actimeters. The influence of harmaline on the mGlu4 mRNA expression in the cerebellum and inferior olive was analysed by in situ hybridization. Harmaline at a dose of 15 mg/kg (ip) triggered tremor which was manifested by an increase in the power within 9-15 Hz band and in the tremor index (a difference in power between bands 9-15 Hz and 0-8 Hz). Harmaline induced a biphasic effect on mobility, initially inhibiting the exploratory locomotor activity of rats (0-30 min after administration), followed by an increase in their basic activity. Lu AF21934 (0.5-5 mg/kg sc) did not influence tremor but at doses of 0.5 and 2.5 mg/kg reversed harmaline-induced hyperactivity. MGlu4 mRNA expression was high in the cerebellar cortex and low in the inferior olive. Repeated harmaline (15 mg/kg ip once a day for 5 days] decreased mGlu4 mRNA in the cerebellum and inferior olive. The present study indicates that the mGlu4 stimulation counteracts hyperactivity induced by harmaline which suggests the involvement of cerebellar glutamatergic transmission in this process. In contrast, neuronal mechanisms involved in tremor seem to be insensitive to the stimulation of mGlu4.

  17. Non-caloric sweeteners, sweetness modulators, and sweetener enhancers.

    PubMed

    DuBois, Grant E; Prakash, Indra

    2012-01-01

    For a new sweetness technology to realize strong commercial success, it must be safe, exhibit good taste quality, be sufficiently soluble and stable in food and beverage systems, and be cost effective and patentable. Assessments of the commercial promise of eight synthetic and eight natural non-caloric sweeteners are made relevant to these metrics. High-potency (HP) non-caloric sweeteners, both synthetic and natural, are generally limited in taste quality by (a) low maximal sweetness response, (b) "off" tastes, (c) slow-onset sweet tastes that linger, and (d) sweet tastes that adapt or desensitize the gustatory system. Formulation approaches to address these limitations are discussed. Enhancement of the normal sucrose sensory response by action of a sweetener receptor positive allosteric modulator (PAM) has been achieved with very significant calorie reduction and with retention of the taste quality of sucrose. Research on PAM discovery over the past decade is summarized.

  18. Neuropharmacological modulation of the P3-like event-related potential in a rat two-tone auditory discrimination task with modafinil and NS9283, a positive allosteric modulator of α4β2 nAChRs.

    PubMed

    Grupe, Morten; Grunnet, Morten; Laursen, Bettina; Bastlund, Jesper Frank

    2014-04-01

    The P300 (P3) event-related potential (ERP) is a neurophysiological signal believed to reflect cognitive processing of salient cues, and is thus used as a measure of attention and working memory. Additionally, P3 amplitude and latency is altered in neurological diseases and can be pharmacologically modulated. As P3-like ERPs can be recorded in rodents, it may serve as a potential translational biomarker of value for drug discovery. Here we investigated whether a positive allosteric modulator of α4β2 nicotinic acetylcholine receptors, NS9283, and the psychostimulant modafinil could modulate P3-like ERPs in healthy adult rats performing an auditory oddball discrimination task. ERPs were recorded with electroencephalography electrodes implanted into mediodorsal (MD) thalamus, medial prefrontal cortex, hippocampus and auditory cortex (AC). P3-like ERPs were detected in all brain regions, displaying larger amplitudes in target trials compared to non-target trials. Administration of modafinil (64 mg/kg) decreased P3-like ERP latency in MD thalamus and AC, whereas NS9283 augmented P3-like ERP amplitude in MD thalamus at 0.3 mg/kg and in AC at 3.0 mg/kg. Additionally, N1 pre-attention peak amplitude in MD thalamus was increased with 0.3 mg/kg NS9283. Neither of the compounds enhanced task performance. Rather, modafinil lowered correct rejections in non-target trials. In summary, our findings reveal pharmacological modulation of the rat P3-like ERP in cortical and subcortical regions by modafinil and NS9283. These findings encourage further exploration of the rat P3-like ERP in order to promote the understanding of its meaning within cognition, as well as its applicability as a translatable biomarker in drug development.

  19. Exploiting protein flexibility to predict the location of allosteric sites

    PubMed Central

    2012-01-01

    Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse

  20. Allosteric modulation of the HIV-1 gp120-gp41 association site by adjacent gp120 variable region 1 (V1) N-glycans linked to neutralization sensitivity.

    PubMed

    Drummer, Heidi E; Hill, Melissa K; Maerz, Anne L; Wood, Stephanie; Ramsland, Paul A; Mak, Johnson; Poumbourios, Pantelis

    2013-01-01

    The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³⁶ in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³⁹INN sequence, which ablates the overlapping Asn¹⁴¹-Asn¹⁴²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu⁵⁹³, Trp⁵⁹⁶ and Lys⁶⁰¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a

  1. Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

    PubMed

    Dias, James A; Campo, Brice; Weaver, Barbara A; Watts, Julie; Kluetzman, Kerri; Thomas, Richard M; Bonnet, Béatrice; Mutel, Vincent; Poli, Sonia M

    2014-01-01

    We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR

  2. Diagnostic modeling of PAMS VOC observation.

    PubMed

    Chen, Sheng-Po; Liu, Tsun-Hsien; Chen, Tu-Fu; Yang, Chang-Feng Ou; Wang, Jia-Lin; Chang, Julius S

    2010-06-15

    Although a number of gas-phase chemical mechanisms, such as CBM-IV, RADM2, and SAPRC have been successful in studying gas-phase atmospheric chemical processes, they all used different combinations of lumped organic species to describe the role of organics in gas-phase chemical processes. Photochemical Assessment Monitoring Stations (PAMS) have been in use for over a decade and yet it is not clear how the detailed organic species measured by PAMS compare to the lumped modeled species. By developing a detailed mechanism specifically for the PAMS organics and embedding this diagnostic model within a regional-scale transport and chemistry model, one can then directly compare PAMS observation with regional-scale model simulations. By means of this comparison one can perhaps better evaluate model performance. The Taiwan Air Quality Model (TAQM) was modified by adding a submodel with transport processes and chemical mechanism for interactions of the 56 species observed by PAMS. It is assumed that TAQM can simulate the overall regional-scale environment including time evolution of oxidants and radicals; these results are then used to simulate the evolution of PAMS organics with species-specific source functions, meteorological transport, and chemical interactions. Model simulations of each PAMS organic were compared with PAMS hourly surface measurements. A case study with data collected at three sites in central Taiwan showed that when meteorological simulations were comparable with observations, diurnal patterns of most organics performed well with PAMS data after emissions were corrected. It is found emissions of over half of the PAMS species require correction, some by surprisingly large factors. With such correlation, simulated time evolution of ratios of ethylbenzene/m,p-xylenes and ethane/n-butane showed similar behaviors as shown by observation data. From the results of PAMS organics diurnal variations as well as indicator ratios, one can conclude that PAMS Air

  3. Pam2 lipopeptides systemically increase myeloid-derived suppressor cells through TLR2 signaling

    SciTech Connect

    Maruyama, Akira; Shime, Hiroaki Takeda, Yohei; Azuma, Masahiro; Matsumoto, Misako; Seya, Tsukasa

    2015-02-13

    Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that exhibit potent immunosuppressive activity. They are increased in tumor-bearing hosts and contribute to tumor development. Toll-like receptors (TLRs) on MDSCs may modulate the tumor-supporting properties of MDSCs through pattern-recognition. Pam2 lipopeptides represented by Pam2CSK4 serve as a TLR2 agonist to exert anti-tumor function by dendritic cell (DC)-priming that leads to NK cell activation and cytotoxic T cell proliferation. On the other hand, TLR2 enhances tumor cell progression/invasion by activating tumor-infiltrating macrophages. How MDSCs respond to TLR2 agonists has not yet been determined. In this study, we found intravenous administration of Pam2CSK4 systemically up-regulated the frequency of MDSCs in EG7 tumor-bearing mice. The frequency of tumor-infiltrating MDSCs was accordingly increased in response to Pam2CSK4. MDSCs were not increased by Pam2CSK4 stimuli in TLR2 knockout (KO) mice. Adoptive transfer experiments using CFSE-labeled MDSCs revealed that the TLR2-positive MDSCs survived long in tumor-bearing mice in response to Pam2CSK4 treatment. Since the increased MDSC population sustained immune-suppressive properties, our study suggests that Pam2CSK4-triggered TLR2 activation enhances the MDSC potential and suppress antitumor immune response in tumor microenvironment. - Highlights: • Pam2CSK4 administration induces systemic accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • TLR2 is essential for Pam2CSK4-induced accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • Pam2CSK4 supports survival of CD11b{sup +}Gr1{sup +} MDSCs in vivo.

  4. The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott–Aldrich syndrome protein

    PubMed Central

    Leung, Daisy W.; Rosen, Michael K.

    2005-01-01

    The GTP/GDP nucleotide switch in Ras superfamily GTPases generally involves differential affinity toward downstream effectors, with the GTP-bound state having a higher affinity for effector than the GDP-bound state. We have developed a quantitative model of allosteric regulation of the Wiskott–Aldrich syndrome protein (WASP) by the Rho GTPase Cdc42 to better understand how GTPase binding is coupled to effector activation. The model accurately predicts WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 as functions of a two-state allosteric equilibrium in WASP. The ratio of GTPase affinities for the inactive and active states of WASP is appreciably larger for Cdc42–GTP than for Cdc42–GDP. The greater ability to distinguish between the two states of WASP makes Cdc42–GTP a full WASP agonist, whereas Cdc42–GDP is only a partial agonist. Thus, the nucleotide switch controls not only the affinity of Cdc42 for its effector but also the efficiency of coupling between the Cdc42-binding and allosteric equilibria in WASP. This effect can ensure high fidelity and specificity in Cdc42 signaling in crowded membrane environments. PMID:15821030

  5. Software system for reducing PAM-2 data

    NASA Technical Reports Server (NTRS)

    Pepin, T. J.

    1982-01-01

    A software system for reducing PAM-II data was constructed. The data reduction process concatenates data tapes; determines ephemeris; and inverts full sun extinction data. Tests of this data reduction process show that PAM-II data can be compared with data from other, similar satellites.

  6. Michigan's PAM Assistance Centre: A Comprehensive Resource.

    ERIC Educational Resources Information Center

    Exceptional Parent, 1983

    1983-01-01

    The PAM (Physically Impaired Association of Michigan) Assistance Center is an information clearinghouse and referral service on arrestive devices begun in l979 and staffed by a multidisciplinary team. PAM provides information on availability, cost, and sources of assistive devices , as well as a center for hands-on displays. (CL)

  7. GPM Rain Rates in Tropical Cyclone Pam

    NASA Video Gallery

    NASA-JAXA's GPM Satellite Close-up of Cyclone Pam's Rainfall NASA-JAXA's GPM core satellite captured rain rates in Tropical Cyclone Pam at 03:51 UTC (2:51 p.m. local time) on March 14, 2015. Heavie...

  8. Discovery of N-(4-methoxy-7-methylbenzo[d]thiazol-2-yl)isonicatinamide, ML293, as a novel, selective and brain penetrant positive allosteric modulator of the muscarinic 4 (M4) receptor

    PubMed Central

    Salovich, James M.; Vinson, Paige N.; Sheffler, Douglas J.; Lamsal, Atin; Utley, Thomas J.; Blobaum, Anna L.; Bridges, Thomas M.; Le, Uyen; Jones, Carrie K.; Wood, Michael R.; Daniels, J. Scott; Conn, P. Jeffrey; Niswender, Colleen M.; Lindsley, Craig W.; Hopkins, Corey R.

    2012-01-01

    Herein we describe the discovery and development of a novel class of M4 positive allosteric modulators, culminating in the discovery of ML293. ML293 exhibited modest potency at the human M4 receptor (EC50 = 1.3 µM) and excellent efficacy as noted by the 14.6-fold leftward shift of the agonist concentration-response curve. ML293 was also selective versus the other muscarinic subtypes and displayed excellent in vivo PK properties in rat with low IV clearance (11.6 mL/min/kg) and excellent brain exposure (PO PBL, 10 mg/kg at 1 h, [Brain] = 10.3 µM, B:P = 0.85). PMID:22738637

  9. A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning.

    PubMed

    Shirey, Jana K; Brady, Ashley E; Jones, Paulianda J; Davis, Albert A; Bridges, Thomas M; Kennedy, J Phillip; Jadhav, Satyawan B; Menon, Usha N; Xiang, Zixiu; Watson, Mona L; Christian, Edward P; Doherty, James J; Quirk, Michael C; Snyder, Dean H; Lah, James J; Levey, Allan I; Nicolle, Michelle M; Lindsley, Craig W; Conn, P Jeffrey

    2009-11-11

    M(1) muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M(1) receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M(1) receptor affinity for acetylcholine. We found that activation of the M(1) receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M(1) receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M(1) receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M(1) receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M(1) mAChR subtype may ameliorate impairments in cognitive function.

  10. Computational Tools for Allosteric Drug Discovery: Site Identification and Focus Library Design.

    PubMed

    Huang, Wenkang; Nussinov, Ruth; Zhang, Jian

    2017-01-01

    Allostery is an intrinsic phenomenon of biological macromolecules involving regulation and/or signal transduction induced by a ligand binding to an allosteric site distinct from a molecule's active site. Allosteric drugs are currently receiving increased attention in drug discovery because drugs that target allosteric sites can provide important advantages over the corresponding orthosteric drugs including specific subtype selectivity within receptor families. Consequently, targeting allosteric sites, instead of orthosteric sites, can reduce drug-related side effects and toxicity. On the down side, allosteric drug discovery can be more challenging than traditional orthosteric drug discovery due to difficulties associated with determining the locations of allosteric sites and designing drugs based on these sites and the need for the allosteric effects to propagate through the structure, reach the ligand binding site and elicit a conformational change. In this study, we present computational tools ranging from the identification of potential allosteric sites to the design of "allosteric-like" modulator libraries. These tools may be particularly useful for allosteric drug discovery.

  11. PAM: A Program for Adolescent Mothers.

    ERIC Educational Resources Information Center

    Robichaux, Faye B.; And Others

    1989-01-01

    Describes the Program for Adolescent Mothers (PAM) established to provide opportunities for teen mothers in Louisiana to increase their self-esteem, become productive citizens, and become aware of the physical and emotional development of children. (JOW)

  12. Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization.

    PubMed

    Wrzal, Paulina K; Devost, Dominic; Pétrin, Darlaine; Goupil, Eugénie; Iorio-Morin, Christian; Laporte, Stéphane A; Zingg, Hans H; Hébert, Terence E

    2012-01-01

    The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.

  13. The Five-To-Six-Coordination Transition of Ferric Human Serum Heme-Albumin Is Allosterically-Modulated by Ibuprofen and Warfarin: A Combined XAS and MD Study

    PubMed Central

    Bionducci, Monica; Fanali, Gabriella; Meli, Massimiliano; Colombo, Giorgio; Fasano, Mauro; Ascenzi, Paolo; Mobilio, Settimio

    2014-01-01

    Human serum albumin (HSA) is involved physiologically in heme scavenging; in turn, heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, the allosteric effect of ibuprofen and warfarin on the local atomic structure around the ferric heme-Fe (heme-Fe(III)) atom of HSA-heme-Fe (HSA-heme-Fe(III)) has been probed by Fe-K edge X-ray absorption spectroscopy (XAS). The quantitative analysis of the Fe-K edge extended X-ray absorption fine structure (EXAFS) signals and modeling of the near edge (XANES) spectral features demonstrated that warfarin and ibuprofen binding modify the local structure of the heme-Fe(III). Combined XAS data analysis and targeted molecular dynamics (MD) simulations provided atomic resolution insights of protein structural rearrangements required to accommodate the heme-Fe(III) upon ibuprofen and warfarin binding. In the absence of drugs, the heme-Fe(III) atom is penta-coordinated having distorted 4+1 configuration made by the nitrogen atoms of the porphyrin ring and the oxygen phenoxy atom of the Tyr161 residue. MD simulations show that ibuprofen and warfarin association to the secondary fatty acid (FA) binding site 2 (FA2) induces a reorientation of domain I of HSA-heme-Fe(III), this leads to the redirection of the His146 residue providing an additional bond to the heme-Fe(III) atom, providing the 5+1 configuration. The comparison of Fe-K edge XANES spectra calculated using MD structures with those obtained experimentally confirms the reliability of the proposed structural model. As a whole, combining XAS and MD simulations it has been possible to provide a reliable model of the heme-Fe(III) atom coordination state and to understand the complex allosteric transition occurring in HSA-heme-Fe(III) upon ibuprofen and warfarin binding. PMID:25153171

  14. State-Dependent Alterations in Sleep/wake Architecture Elicited by the M4 PAM VU0467154- Relation to Antipsychotic-like Drug Effects

    PubMed Central

    Gould, Robert W.; Nedelcovych, Michael T.; Gong, Xuewen; Tsai, Erica; Bubser, Michael; Bridges, Thomas M.; Wood, Michael R.; Duggan, Mark E.; Brandon, Nicholas J.; Dunlop, John; Wood, Michael W.; Ivarsson, Magnus; Noetzel, Meredith J.; Daniels, J. Scott; Niswender, Colleen M.; Lindsley, Craig W.; Conn, P. Jeffrey; Jones, Carrie K.

    2015-01-01

    Accumulating evidence indicates direct relationships between sleep abnormalities and the severity and prevalence of other symptom clusters in schizophrenia. Assessment of potential state-dependent alterations in sleep architecture and arousal relative to antipsychotic-like activity is critical for the development of novel antipsychotic drugs (APDs). Recently, we reported that VU0467154, a selective positive allosteric modulator (PAM) of the M4 muscarinic acetylcholine receptor (mAChR), exhibits robust APD-like and cognitive enhancing activity in rodents. However, the state-dependent effects of VU0467154 on sleep architecture and arousal have not been examined. Using polysomnography and quantitative electroencephalographic recordings from subcranial electrodes in rats, we evaluated the effects of VU0467154, in comparison with the atypical APD clozapine and the M1/M4-preferring mAChR agonist xanomeline. VU0467154 induced state-dependent alterations in sleep architecture and arousal by delaying Rapid Eye Movement (REM) sleep onset, selectively increased cumulative duration of total and Non-Rapid Eye Movement (NREM) sleep, and increased arousal during waking periods. Clozapine decreased arousal during wake, increased cumulative NREM, and decreased REM sleep. In contrast, xanomeline increased time awake and arousal during wake, but reduced slow wave activity during NREM sleep. Additionally, in combination with the N-methyl-d-aspartate subtype of glutamate receptor (NMDAR) antagonist MK-801, modeling NMDAR hypofunction thought to underlie many symptoms in schizophrenia, both VU0467154 and clozapine attenuated MK-801-induced elevations in high frequency gamma power consistent with an APD-like mechanism of action. These findings suggest that selective M4 PAMs may represent a novel mechanism for treating multiple symptoms of schizophrenia, including disruptions in sleep architecture without a sedative profile. PMID:26617071

  15. State-dependent alterations in sleep/wake architecture elicited by the M4 PAM VU0467154 - Relation to antipsychotic-like drug effects.

    PubMed

    Gould, Robert W; Nedelcovych, Michael T; Gong, Xuewen; Tsai, Erica; Bubser, Michael; Bridges, Thomas M; Wood, Michael R; Duggan, Mark E; Brandon, Nicholas J; Dunlop, John; Wood, Michael W; Ivarsson, Magnus; Noetzel, Meredith J; Daniels, J Scott; Niswender, Colleen M; Lindsley, Craig W; Conn, P Jeffrey; Jones, Carrie K

    2016-03-01

    Accumulating evidence indicates direct relationships between sleep abnormalities and the severity and prevalence of other symptom clusters in schizophrenia. Assessment of potential state-dependent alterations in sleep architecture and arousal relative to antipsychotic-like activity is critical for the development of novel antipsychotic drugs (APDs). Recently, we reported that VU0467154, a selective positive allosteric modulator (PAM) of the M4 muscarinic acetylcholine receptor (mAChR), exhibits robust APD-like and cognitive enhancing activity in rodents. However, the state-dependent effects of VU0467154 on sleep architecture and arousal have not been examined. Using polysomnography and quantitative electroencephalographic recordings from subcranial electrodes in rats, we evaluated the effects of VU0467154, in comparison with the atypical APD clozapine and the M1/M4-preferring mAChR agonist xanomeline. VU0467154 induced state-dependent alterations in sleep architecture and arousal including delayed Rapid Eye Movement (REM) sleep onset, increased cumulative duration of total and Non-Rapid Eye Movement (NREM) sleep, and increased arousal during waking periods. Clozapine decreased arousal during wake, increased cumulative NREM, and decreased REM sleep. In contrast, xanomeline increased time awake and arousal during wake, but reduced slow wave activity during NREM sleep. Additionally, in combination with the N-methyl-d-aspartate subtype of glutamate receptor (NMDAR) antagonist MK-801, modeling NMDAR hypofunction thought to underlie many symptoms in schizophrenia, both VU0467154 and clozapine attenuated MK-801-induced elevations in high frequency gamma power consistent with an APD-like mechanism of action. These findings suggest that selective M4 PAMs may represent a novel mechanism for treating multiple symptoms of schizophrenia, including disruptions in sleep architecture without a sedative profile.

  16. Joint carrier phase and symbol timing recovery for PAM systems

    NASA Astrophysics Data System (ADS)

    Meyers, M. H.; Franks, L. E.

    1980-08-01

    The theory of maximum likelihood (ML) estimation as applied to PAM timing and phase recovery is considered. Data-aided (DA) and nondata-aided (NDA) strategies used for the joint estimation of both phase and timing parameters are evaluated on the basis of their error variances. The comparisons of the effects of excess bandwidth, different modulation schemes, DA versus NDA recovery, and joint estimation versus estimation of only one parameter are presented. A practical implementation of a proposed ML estimator, named a pseudo-maximum likelihood (PML) estimator, exhibits a noise-independent, data dependent jitter that dominates in many cases of practical interest.

  17. Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

    PubMed

    Martin, Brandon S; Martinez-Botella, Gabriel; Loya, Carlos M; Salituro, Francesco G; Robichaud, Albert J; Huntsman, Molly M; Ackley, Mike A; Doherty, James J; Corbin, Joshua G

    2016-06-01

    Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

  18. Tropical cyclone Pam field survey in Vanuatu

    NASA Astrophysics Data System (ADS)

    Fritz, Hermann M.; Pilarczyk, Jessica E.; Kosciuch, Thomas; Hong, Isabel; Rarai, Allan; Harrison, Morris J.; Jockley, Fred R.; Horton, Benjamin P.

    2016-04-01

    Severe tropical cyclone Pam (Cat. 5, SSHS) crossed the Vanuatu archipelago with sustained winds of 270 km/h on March 13 and 14, 2015 and made landfall on Erromango. Pam is the most intense tropical cyclone to make landfall on Vanuatu since the advent of satellite imagery based intensity estimates in the 1970s. Pam caused one of the worst natural disaster in Vanuatu's recorded history. Eleven fatalities were directly attributed to cyclone Pam and mostly due to lack of shelter from airborne debris. On March 6 Pam formed east of the Santa Cruz Islands causing coastal inundation on Tuvalu's Vaitupu Island located some 1100 km east of the cyclone center. Pam intensified while tracking southward along Vanuatu severely affecting the Shefa and Tafea Provinces. An international storm surge reconnaissance team was deployed to Vanuatu from June 3 to 17, 2015 to complement earlier local surveys. Cyclone Pam struck a remote island archipelago particularly vulnerable to the combined cyclonic multi-hazards encompassing extreme wind gusts, massive rainfall and coastal flooding due to a combination of storm surge and storm wave impacts. The team surveyed coastal villages on Epi, the Shepherd Islands (Tongoa and Mataso), Efate (including Lelepa), Erromango, and Tanna. The survey spanned 320 km parallel to the cyclone track between Epi and Tanna encompassing more than 45 sites including the hardest hit settlements. Coastal flooding profiles were surveyed from the shoreline to the limit of inundation. Maximum coastal flood elevations and overland flow depths were measured based on water marks on buildings, scars on trees, rafted debris and corroborated with eyewitness accounts. We surveyed 91 high water marks with characteristic coastal flood levels in the 3 to 7 m range and composed of storm surge with superimposed storm waves. Inundation distances were mostly limited to a few hundred meters but reached 800 m on Epi Island. Wrack lines containing pumice perfectly delineated the

  19. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein

    PubMed Central

    Karpowicz, Przemysław; Osmulski, Paweł A.; Witkowska, Julia; Sikorska, Emilia; Giżyńska, Małgorzata; Belczyk-Ciesielska, Agnieszka; Gaczynska, Maria E.; Jankowska, Elżbieta

    2015-01-01

    The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to significantly improve the inhibitory potency. PMID:26575189

  20. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface*

    PubMed Central

    Jayakar, Selwyn S.; Zhou, Xiaojuan; Savechenkov, Pavel Y.; Chiara, David C.; Desai, Rooma; Bruzik, Karol S.; Miller, Keith W.; Cohen, Jonathan B.

    2015-01-01

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ+-β− subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β+-α− subunit interfaces. GABA inhibits S-[3H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[3H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ+-β− site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. PMID:26229099

  1. Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin-1 receptor, by positional scanning using β-amino γ-lactams.

    PubMed

    Boutard, Nicolas; Turcotte, Stéphane; Beauregard, Kim; Quiniou, Christiane; Chemtob, Sylvain; Lubell, William D

    2011-04-01

    The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin-1 receptor 101.10 (D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH₂) has been studied using (R)- and (S)-Bgl residues. Twelve Bgl peptides were synthesized using (R)- and (S)-cyclic sulfamidate reagents derived from L- and D-aspartic acid in an optimized Fmoc-compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)- and (S)-Bgl 101.10 analogs for their potential to inhibit IL-1β-induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II'β-turn-like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity.

  2. Discovery and Preclinical Characterization of 3-((4-(4-Chlorophenyl)-7-fluoroquinoline-3-yl)sulfonyl)benzonitrile, a Novel Non-acetylenic Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulator for Psychiatric Indications.

    PubMed

    Galambos, János; Bielik, Attila; Krasavin, Mikhail; Orgován, Zoltán; Domány, György; Nógrádi, Katalin; Wágner, Gábor; Balogh, György T; Béni, Zoltán; Kóti, János; Szakács, Zoltán; Bobok, Amrita; Kolok, Sándor; Mikó-Bakk, Mónika L; Vastag, Mónika; Sághy, Katalin; Laszy, Judit; Halász, Attila Sándor; Balázs, Ottilia; Gál, Krisztina; Greiner, István; Szombathelyi, Zsolt; Keserű, György M

    2017-03-23

    Negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGluR5) have been implicated as a potential pharmacotherapy for a number of psychiatric diseases, including anxiety and depression. Most of the mGluR5 NAM clinical candidates can be characterized by the central acetylenic moiety that connects the terminal pharmacophores. Identification of a sulfoquinoline hit via high throughput screening (HTS) followed by optimization provided a 4-phenyl-3-aryl-sulfoquinoline lead compound with the minimal pharmacophore. Optimization of the core and aryl appendages was performed by scanning and matrix libraries synthesized by the multiple parallel synthesis approach. Biological evaluation of matrix libraries provided a number of potent, metabolically stable, and in vivo active compounds. One of these compounds, 25 showed high efficacy and safety in preclinical in vivo models; this allowed its nomination as a novel, nonacetylenic mGluR5 NAM clinical candidate. Compound 25 was advanced to first-in-man trials for the treatment of psychiatric conditions.

  3. Study on RZ-4PAM downstream signals with duty cycles of 33% and 50% for optical access system application

    NASA Astrophysics Data System (ADS)

    Chen, Fu-ping; Shao, Yu-feng; Chen, Lao; Shen, Shi-lu; Wang, Anrong; Luo, Ying-xiang; Zhao, Yun-jie

    2017-01-01

    4-ary pulse amplitude modulation (4PAM) signals with 33% and 50% return-to-zero (RZ) clocks are generated for passive optical network (PON). We demonstrate that RZ-4PAM signals with duty cycles of 33% and 50% after transmission over 20-km-long single mode fiber (SMF) at 10 Gbit/s can be directly detected by using one photo detector, and the original data can also be restored by one M-ary threshold detector and one 4PAM sequence decoder. The optical spectra of 33% and 50% RZ-4PAM signals are measured, and their eye-diagrams before and after transmission are also analyzed. Simulation results show that 33% and 50% RZ-4PAM downlink signals can be received effectively, and the received power values are -15.1 dBm and -13.8 dBm when the bit error rate (BER) is 10-6. Moreover, 33% RZ-4PAM optical signals have better reception performance than 50% RZ-4PAM optical signals.

  4. USDA-ARS perspective on PAM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyacrylamide (PAM) described herein is a synthetic organic polymer used globally by a number of important industries. It also has a number of valuable applications in irrigated agriculture, including its use in furrow irrigation to control erosion and sediment loss in runoff, manage infiltration,...

  5. Allosteric dynamics of SAMHD1 studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Patra, K. K.; Bhattacharya, A.; Bhattacharya, S.

    2016-10-01

    SAMHD1 is a human cellular enzyme that blocks HIV-1 infection in myeloid cells and non-cycling CD4+T cells. The enzyme is an allosterically regulated triphosphohydrolase that modulates the level of cellular dNTP. The virus restriction is attributed to the lowering of the pool of dNTP in the cell to a point where reverse-transcription is impaired. Mutations in SAMHD1 are also implicated in Aicardi-Goutieres syndrome. A mechanistic understanding of the allosteric activation of the enzyme is still elusive. We have performed molecular dynamics simulations to examine the allosteric site dynamics of the protein and to examine the connection between the stability of the tetrameric complex and the Allosite occupancy.

  6. OFDM and PAM comparison using a high baudrate low resolution IM/DD interface for 400G Ethernet access.

    PubMed

    André, Nuno Sequeira; Louchet, Hadrien; Filsinger, Volker; Hansen, Erik; Richter, André

    2016-05-30

    We compare OFDM and PAM for 400G Ethernet based on a 3-bit high baudrate IM/DD interface at 1550nm. We demonstrate 27Gb/s and 32Gb/s transmission over 10km SSMF using OFDM and PAM respectively. We show that capacity can be improved through adaptation/equalization to achieve 42Gb/s and 64Gb/s for OFDM and PAM respectively. Experimental results are used to create realistic simulations to extrapolate the performance of both modulation formats under varied conditions. For the considered interface we found that PAM has the best performance, OFDM is impaired by quantization noise. When the resolution limitation is relaxed, OFDM shows better performance.

  7. Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice

    PubMed Central

    Tracy, Matthew E.; Banks, Matthew L.; Shelton, Keith L.

    2016-01-01

    Rationale There is an emerging body of evidence that implicates a crucial role of γ-aminobutyric acid subtype A (GABAA) receptors in modulating the rewarding effects of a number of abused drugs. Modulation of GABAA receptors may therefore represent a novel drug-class independent mechanism for the development of abuse treatment pharmacotherapeutics. Objectives We tested the hypothesis that the GABAA receptor benzodiazepine-site (BDZ) negative modulator Ro15-4513 would reduce the reward-related effects of three pharmacologically dissimilar drugs; toluene vapor, d-methamphetamine and diazepam using intracranial self-stimulation (ICSS) in mice. We also examined whether Ro15-4513 attenuated dopamine release produced by d-methamphetamine in an in vivo microdialysis procedure. Results Ro15-4513 abolished ICSS reward facilitation produced by all three abused drugs at Ro15-4513 doses which had no effect on ICSS when administered alone. In contrast, the BDZ antagonist flumazenil only attenuated the ICSS-facilitating effects of diazepam. Administration of the same dose of Ro15-4513 which abolished drug-facilitated ICSS produced a 58% decrease in d-methamphetamine stimulated dopamine in the nucleus accumbens of mice relative to d-methamphetamine alone. Conclusions These results demonstrate that negative modulation of GABAA receptors can produce profound reductions in reward-related effects on a diverse group of drugs that activate the mesolimbic reward pathway through different mechanisms. These data suggest that pharmacological modulation of GABAA receptors may represent a viable pathway for the development of drug abuse pharmacotherapies. PMID:26612620

  8. Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators

    PubMed Central

    Rajamohan, Francis; Reyes, Allan R.; Frisbie, Richard K.; Hoth, Lise R.; Sahasrabudhe, Parag; Magyar, Rachelle; Landro, James A.; Withka, Jane M.; Caspers, Nicole L.; Calabrese, Matthew F.; Ward, Jessica; Kurumbail, Ravi G.

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with β1-containing heterotrimers, β2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases. PMID:26635351

  9. Use of a novel rapid and resource-efficient cassette dosing approach to determine the pharmacokinetics and CNS distribution of small molecule 7-transmembrane receptor allosteric modulators in rat

    PubMed Central

    Bridges, Thomas M; Morrison, Ryan D; Byers, Frank W; Luo, Shuanghui; Scott Daniels, J

    2014-01-01

    Approaches to efficiently and accurately define the pharmacokinetics (PK) of large sets of small molecules in rodents have been previously described. Likewise, a variety of methods for determining brain tissue distribution (BTD) have been reported for use in the discovery of therapeutics targeting the central nervous system (CNS). Herein we describe a novel cassette approach to efficiently obtain concurrent PK and BTD data from a dose of up to five compounds in one rat over 24 h. In conjunction with fraction unbound (fu) data obtained in plasma and brain homogenate, this approach serves as an efficient means to determine compound unbound brain:unbound plasma partition coefficients (Kp,uu), thereby providing insight to compounds bearing poor permeability and/or active transporter activity impacting their permeation of the blood–brain barrier (BBB). This integrated approach was utilized in a lead optimization effort towards the discovery of CNS-penetrant allosteric modulators of a seven-transmembrane (7TM) receptor target. Rat PK and brain distribution was rapidly obtained for 70 compounds and correlated to data obtained from in vitro assessments. Two compounds that were evaluated in cassette and discrete studies, displayed agreement in PK (compound 1: cassette CLp = 1.6 mL min−1 kg−1, discrete CLp = 1.6 mL min−1 kg−1; compound 2: cassette CLp = 11 mL min−1 kg−1, discrete CLp = 8.1 mL min−1 kg−1) and BTD (compound 1: cassette Kp = 0.11, discrete Kp = 0.09; compound 2: cassette Kp < 0.05, discrete Kp = 0.04). The resulting data were used to guide medicinal chemistry efforts and to enable the progression of optimized compounds to in vivo pharmacodynamic assessments. PMID:25505618

  10. A Negative Allosteric Modulator for α5 Subunit-Containing GABA Receptors Exerts a Rapid and Persistent Antidepressant-Like Action without the Side Effects of the NMDA Receptor Antagonist Ketamine in Mice

    PubMed Central

    Nelson, Mackenzie E.; Krimmel, Samuel R.; Georgiou, Polymnia; Gould, Todd D.

    2017-01-01

    Abstract New antidepressant pharmacotherapies that provide rapid relief of depressive symptoms are needed. The NMDA receptor antagonist ketamine exerts rapid antidepressant actions in depressed patients but also side effects that complicate its clinical utility. Ketamine promotes excitatory synaptic strength, likely by producing high-frequency correlated activity in mood-relevant regions of the forebrain. Negative allosteric modulators of GABA-A receptors containing α5 subunits (α5 GABA-NAMs) should also promote high-frequency correlated electroencephalogram (EEG) activity and should therefore exert rapid antidepressant responses. Because α5 subunits display a restricted expression in the forebrain, we predicted that α5 GABA-NAMs would produce activation of principle neurons but exert fewer side effects than ketamine. We tested this hypothesis in male mice and observed that the α5 GABA-NAM MRK-016 exerted an antidepressant-like response in the forced swim test at 1 and 24 h after administration and an anti-anhedonic response after chronic stress in the female urine sniffing test (FUST). Like ketamine, MRK-016 produced a transient increase in EEG γ power, and both the increase in γ power and its antidepressant effects in the forced swim test were blocked by prior administration of the AMPA-type glutamate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). Unlike ketamine, however, MRK-016 produced no impairment of rota-rod performance, no reduction of prepulse inhibition (PPI), no conditioned-place preference (CPP), and no change in locomotion. α5 GABA-NAMs, thus reproduce the rapid antidepressant-like actions of ketamine, perhaps via an AMPA receptor (AMPAR)-dependent increase in coherent neuronal activity, but display fewer potential negative side effects. These compounds thus demonstrate promise as clinically useful fast-acting antidepressants. PMID:28275719

  11. [G-protein-coupled receptors targeting: the allosteric approach].

    PubMed

    Sebag, Julien A; Pantel, Jacques

    2012-10-01

    G-protein-coupled receptors (GPCR) are a major family of drug targets. Essentially all drugs targeting these receptors on the market compete with the endogenous ligand (agonists or antagonists) for binding the receptor. Recently, non-competitive compounds binding to distinct sites from the cognate ligand were documented in various classes of these receptors. These compounds, called allosteric modulators, generally endowed of a better selectivity are able to modulate specifically the endogenous signaling of the receptor. To better understand the promising potential of this class of GPCRs targeting compounds, this review highlights the properties of allosteric modulators, the strategies used to identify them and the challenges associated with the development of these compounds.

  12. Generation-3 programmable array microscope (PAM) with digital micro-mirror device (DMD)

    NASA Astrophysics Data System (ADS)

    De Beule, Pieter A. A.; de Vries, Anthony H. B.; Arndt-Jovin, Donna J.; Jovin, Thomas M.

    2011-03-01

    We report progress on the construction of an optical sectioning programmable array microscope (PAM) implemented with a digital micro-mirror device (DMD) spatial light modulator (SLM) utilized for both fluorescence illumination and detection. The introduction of binary intensity modulation at the focal plane of a microscope objective in a computer controlled pixilated mode allows the recovery of an optically sectioned image. Illumination patterns can be changed very quickly, in contrast to static Nipkow disk or aperture correlation implementations, thereby creating an optical system that can be optimized to the optical specimen in a convenient manner, e.g. for patterned photobleaching, photobleaching reduction, or spatial superresolution. We present a third generation (Gen-3) dual path PAM module incorporating the 25 kHz binary frame rate TI 1080p DMD and a newly developed optical system that offers diffraction limited imaging with compensation of tilt angle distortion.

  13. EEG-β/γ spectral power elevation in rat: a translatable biomarker elicited by GABA(Aα2/3)-positive allosteric modulators at nonsedating anxiolytic doses.

    PubMed

    Christian, Edward P; Snyder, Dean H; Song, Wei; Gurley, David A; Smolka, Joanne; Maier, Donna L; Ding, Min; Gharahdaghi, Farzin; Liu, Xiaodong F; Chopra, Maninder; Ribadeneira, Maria; Chapdelaine, Marc J; Dudley, Adam; Arriza, Jeffrey L; Maciag, Carla; Quirk, Michael C; Doherty, James J

    2015-01-01

    Benzodiazepine drugs, through interaction with GABA(Aα1), GABA(Aα2,3), and GABA(Aα5) subunits, modulate cortical network oscillations, as reflected by a complex signature in the EEG power spectrum. Recent drug discovery efforts have developed GABA(Aα2,3)-subunit-selective partial modulators in an effort to dissociate the side effect liabilities from the efficacy imparted by benzodiazepines. Here, we evaluated rat EEG and behavioral end points during dosing of nine chemically distinct compounds that we confirmed statistically to selectively to enhance GABA(Aα2,3)-mediated vs. GABA(Aα1) or GABA(Aα5) currents in voltage clamped oocytes transfected with those GABA(A) subunits. These compounds were shown with in vivo receptor occupancy techniques to competitively displace [(3)H]flumazenil in multiple brain regions following peripheral administration at increasing doses. Over the same dose range, the compounds all produced dose-dependent EEG spectral power increases in the β- and and γ-bands. Finally, the dose range that increased γ-power coincided with that eliciting punished over unpunished responding in a behavioral conflict model of anxiety, indicative of anxiolysis without sedation. EEG γ-band power increases showed a significant positive correlation to in vitro GABA(Aα2,3) modulatory intrinsic activity across the compound set, further supporting a hypothesis that this EEG signature was linked specifically to pharmacological modulation of GABA(Aα2,3) signaling. These findings encourage further evaluation of this EEG signature as a noninvasive clinical translational biomarker that could ultimately facilitate development of GABA(Aα2,3)-subtype-selective drugs for anxiety and potentially other indications.

  14. ASBench: benchmarking sets for allosteric discovery.

    PubMed

    Huang, Wenkang; Wang, Guanqiao; Shen, Qiancheng; Liu, Xinyi; Lu, Shaoyong; Geng, Lv; Huang, Zhimin; Zhang, Jian

    2015-08-01

    Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

  15. Studying the binding interactions of allosteric agonists and antagonists of the CXCR4 receptor.

    PubMed

    Planesas, Jesús M; Pérez-Nueno, Violeta I; Borrell, José I; Teixidó, Jordi

    2015-07-01

    Several examples of allosteric modulators of GPCRs have been reported recently in the literature, but understanding their molecular mechanism presents a new challenge for medicinal chemistry. For the specific case of the cellular receptor CXCR4, it is known that pepducins (lipidated fragments of intracellular GPCR loops) such as ATI-2341 modulate CXCR4 activity agonistically via an allosteric mechanism. Moreover, there are also examples of small organic molecules such as AMD11070 and GSK812397 which may also act as allosteric antagonists. However, incomplete knowledge of the ligand-binding sites has hampered a detailed molecular understanding of how these inhibitors work. Here, we attempt to answer this question by analysing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose two different allosteric binding sites, one located in the intracellular loops 1, 2 and 3 (ICL1, ICL2 and ICL3) which binds the pepducin agonist ATI-2341, and the other at a subsite of the main extracellular orthosteric binding pocket between extracellular loops 1 and 2 and the N-terminus, which binds the antagonists AMD11070 and GSK812397. Allosteric interactions between the CXCR4 and ATI-2341 were predicted by combining different modeling approaches. First, a rotational blind docking search was applied and the best poses were subsequently refined using flexible docking methods and molecular dynamic simulations. For the AMD11070 and GSK812397 antagonists, the entire CXCR4 protein surface was explored by blind docking in order to define the binding region. A second docking analysis by subsites was then performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 allosteric modulators.

  16. Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors

    PubMed Central

    Malherbe, P; Masciadri, R; Norcross, R D; Knoflach, F; Kratzeisen, C; Zenner, M-T; Kolb, Y; Marcuz, A; Huwyler, J; Nakagawa, T; Porter, R H P; Thomas, A W; Wettstein, J G; Sleight, A J; Spooren, W; Prinssen, E P

    2008-01-01

    Background and purpose: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors. Experimental approach: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Gα16-hGABAB(1a,2a) cells by Fluorometric Imaging Plate Reader and GTPγ[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models. Key results: In GTPγ[35S]-binding assays, 0.3 μM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration–response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg−1 p.o. rac-BHFF (100 mg kg−1 p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. Conclusions and implications: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems. PMID:18536733

  17. Estimating Cognitive Profiles Using Profile Analysis via Multidimensional Scaling (PAMS).

    PubMed

    Kim, Se-Kang; Frisby, Craig L; Davison, Mark L

    2004-10-01

    Two of the most popular methods of profile analysis, cluster analysis and modal profile analysis, have limitations. First, neither technique is adequate when the sample size is large. Second, neither method will necessarily provide profile information in terms of both level and pattern. A new method of profile analysis, called Profile Analysis via Multidimensional Scaling (PAMS; Davison, 1996), is introduced to meet the challenge. PAMS extends the use of simple multidimensional scaling methods to identify latent profiles in a multi-test battery. Application of PAMS to profile analysis is described. The PAMS model is then used to identify latent profiles from a subgroup (N = 357) within the sample of the Woodcock-Johnson Psychoeducational Battery-Revised (WJ-R; McGrew, Werder, & Woodcock, 1991; Woodcock & Johnson, 1989), followed by a discussion of procedures for interpreting participants' observed score profiles from the latent PAMS profiles. Finally, advantages and limitations of the PAMS technique are discussed.

  18. Scaling single-wavelength optical interconnects to 180 Gb/s with PAM-M and pulse shaping

    NASA Astrophysics Data System (ADS)

    Dris, Stefanos; Bakopoulos, Paraskevas; Argyris, Nikolaos; Spatharakis, Christos; Avramopoulos, Hercules

    2016-03-01

    Faced with surging datacenter traffic demand, system designers are turning to multi-level optical modulation with direct detection as the means of reaching 100 Gb/s in a single optical lane; a further upgrade to 400 Gb/s is envisaged through wavelength-multiplexing of multiple 100 Gb/s strands. In terms of modulation formats, PAM-4 and PAM-8 are considered the front-runners, striking a good balance between bandwidth-efficiency and implementation complexity. In addition, the emergence of energy-efficient, high-speed CMOS digital-to-analog converters (DACs) opens up new possibilities: Spectral shaping through digital filtering will allow squeezing even more data through low-cost, low-bandwidth electro-optic components. In this work we demonstrate an optical interconnect based on an EAM that is driven directly with sub-volt electrical swing by a 65 GSa/s arbitrary waveform generator (AWG). Low-voltage drive is particularly attractive since it allows direct interfacing with the switch/server ASIC, eliminating the need for dedicated, power-hungry and expensive electrical drivers. Single-wavelength throughputs of 180 and 120 Gb/s are experimentally demonstrated with 60 Gbaud optical PAM-8 and PAM-4 respectively. Successful transmission over 1250 m SMF is achieved with direct-detection, using linear equalization via offline digital signal processing in order to overcome the strong bandwidth limitation of the overall link (~20 GHz). The suitability of Nyquist pulse shaping for optical interconnects is also investigated experimentally with PAM-4 and PAM-8, at a lower symbol rate of 40 Gbaud (limited by the sampling rate of the AWG). To the best of our knowledge, the rates achieved are the highest ever using optical PAM-M formats.

  19. Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

    NASA Astrophysics Data System (ADS)

    Hiraki, Masahiko; Watanabe, Shokei; Chavas, Leonard M. G.; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Wakatsuki, Soichi; Fujihashi, Masahiro; Miki, Kunio; Baba, Seiki; Ueno, Go; Yamamoto, Masaki; Suzuki, Mamoru; Nakagawa, Atsushi; Watanabe, Nobuhisa; Tanaka, Isao

    2010-06-01

    To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

  20. Intraoperative surgical photoacoustic microscopy (IS-PAM) using augmented reality

    NASA Astrophysics Data System (ADS)

    Lee, Changho; Han, Seunghoon; Kim, Sehui; Jeon, Mansik; Kim, Jeehyun; Kim, Chulhong

    2014-03-01

    We have developed an intraoperative surgical photoacoustic microscopy (IS-PAM) system by integrating an optical resolution photoacoustic microscopy (OR-PAM) and conventional surgical microscope. Based on the common optical path in the OR-PAM and microscope system, we can acquire the PAM and microscope images at the same time. Furthermore, by utilizing a mini-sized beam projector, 2D PAM images are back-projected onto the microscope view plane as augmented reality. Thus, both the conventional microscopic and 2D cross-sectional PAM images are displayed on the plane through an eyepiece lens of the microscope. In our method, additional image display tool is not required to show the PAM image. Therefore, it potentially offers significant convenience to surgeons without movement of their sights during surgeries. In order to demonstrate the performance of our IS-PAM system, first, we successfully monitored needle intervention in phantoms. Moreover, we successfully guided needle insertion into mice skins in vivo by visualizing surrounding blood vessels from the PAM images and the magnified skin surfaces from the conventional microscopic images simultaneously.

  1. Allosteric regulation of phenylalanine hydroxylase.

    PubMed

    Fitzpatrick, Paul F

    2012-03-15

    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  2. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  3. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  4. 2-Adic clustering of the PAM matrix.

    PubMed

    Khrennikov, A Yu; Kozyrev, S V

    2009-12-07

    In this paper we demonstrate that the use of the system of 2-adic numbers provides a new insight to some problems of genetics, in particular, degeneracy of the genetic code and the structure of the PAM matrix in bioinformatics. The 2-adic distance is an ultrametric and applications of ultrametric in bioinformatics are not surprising. However, by using the 2-adic numbers we match ultrametric with a number theoretic structure. In this way we find new applications of an ultrametric which differ from known up to now in bioinformatics. We obtain the following results. We show that the PAM matrix A allows the expansion into the sum of the two matrices A=A((2))+A((infinity)), where the matrix A((2)) is 2-adically regular (i.e. matrix elements of this matrix are close to locally constant with respect to the discussed earlier by the authors 2-adic parametrization of the genetic code), and the matrix A((infinity)) is sparse. We discuss the structure of the matrix A((infinity)) in relation to the side chain properties of the corresponding amino acids. We introduce the family of substitution matrices A(alpha,beta)=alpha A((2))+beta A((infinity)), alpha,beta>or=0 which should allow to vary the alignment procedure in order to take into account the different chemical and geometric properties of the amino acids.

  5. Biased signalling and allosteric machines: new vistas and challenges for drug discovery.

    PubMed

    Kenakin, Terry P

    2012-03-01

    Seven transmembrane receptors (7TMRs) are nature's prototype allosteric proteins made to bind molecules at one location to subsequently change their shape to affect the binding of another molecule at another location. This paper attempts to describe the divergent 7TMR behaviours (i.e. third party allostery, receptor oligomerization, biased agonism) observed in pharmacology in terms of a homogeneous group of allosteric behaviours. By considering the bodies involved as a vector defined by a modulator, conduit and guest, these activities can all be described by a simple model of functional allostery made up of the Ehlert allosteric model and the Black/Leff operational model. It will be shown how this model yields parameters that can be used to characterize the activity of any ligand or protein producing effect through allosteric interaction with a 7TMR.

  6. Allosteric Optical Control of a Class B G‐Protein‐Coupled Receptor

    PubMed Central

    Broichhagen, Johannes; Johnston, Natalie R.; von Ohlen, Yorrick; Meyer‐Berg, Helena; Jones, Ben J.; Bloom, Stephen R.; Rutter, Guy A.

    2016-01-01

    Abstract Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca2+, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R. PMID:27059784

  7. Community-level microalgal toxicity assessment by multiwavelength-excitation PAM fluorometry.

    PubMed

    Schmitt-Jansen, Mechthild; Altenburger, Rolf

    2008-01-20

    In ecotoxicological studies involving community-level investigations, rapid and multiparametric fluorescence-based methods may provide substantial advantages over traditional methods used for structural and functional community analysis. Therefore, multiwavelength-excitation pulse-amplitude modulated (PAM) fluorometry was applied in this study to assess long-term changes in periphyton community structure, short-term effects on periphyton functioning (photosynthesis) and pollution induced community tolerance (PICT). For inter-calibration, periphyton structure was evaluated by chemotaxonomic analysis of accessory pigments and a four-wavelength-excitation PAM fluorometer. Short-term effects of herbicides were evaluated by fluorescence quenching analysis and (14)C-incorporation as a proxy of primary production. Subsequently, the method was applied to assess structural and functional changes in periphyton communities after isoproturon exposure for 14 and 26 days, respectively. Results showed good correlation of the PAM fluorescence-based measurements with traditional methods for biofilms in the initial colonisation phase for structural and functional parameters. However, for biofilms older than 9 weeks PAM fluorescence may underestimate biomass. Multiwavelength-excitation PAM fluorometry showed good correlation with marker pigment concentrations indicating that this method provides a reliable estimate of the community structure. PAM fluorometry was able to quantify changes of biomass and follow relative shifts in class composition of biofilms under exposure of isoproturon. Short-term tests based on the quantification of the inhibition of the effective quantum yield revealed a concentration-dependent increase of PICT. The observation of two succession phases of the biofilms after 14 and 26 days of growth, respectively, revealed that sensitivity of biofilms decreased with increasing age and biomass, respectively, but PICT remained a characteristic parameter of exposed

  8. Feasibility of obtaining GPS/PAM-DII telemetry from PKM ignition through PAM-DII separation

    NASA Astrophysics Data System (ADS)

    Friedlander, M. M.; Winn, B. E.; Yang, J. L.; Shiokari, T.

    1986-10-01

    The Shuttle version of the NAVSTAR Block II Global Positioning System (GPS) Space Vehicle (SV) will be injected into the transfer orbit with a Perigee Kick Motor (PKM) contained in an attached PAM-DII stage. Mission 'drift' orbit injection will occur near the 4th apogee of the transfer orbit with an Apogee Kick Motor (AKM) burn. Final mission orbit will be attained following any necessary corrections by the Reaction Control System (RCS) which will also be used throughout the SV lifetime for orbit maintenance. A primary concern in the transfer procedure is GPS/PAM-DII separation following PKM burnout because of the possibility of SV and PAM-DII recontact if large coning angles have developed during the burn. Hence, initial flights will need substantial instrumentation to monitor PKM burn and separation data. The most desirable method of instrumentation readout is by telemetry transmission to an Air Force Remote Tracking station (RTS). This paper shows that the PKM burn can be planned to occur within the field of view of an RTS within the desired look angle constraints. A detailed example and an extensive series of mission planning charts are also provided.

  9. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    NASA Astrophysics Data System (ADS)

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

  10. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-05

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action.

  11. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    PubMed Central

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors. PMID:26823026

  12. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  13. Diagnostic Modeling of PAMS VOC Observation on Regional Scale Environment

    NASA Astrophysics Data System (ADS)

    Chen, S.; Liu, T.; Chen, T.; Ou Yang, C.; Wang, J.; Chang, J. S.

    2008-12-01

    While a number of gas-phase chemical mechanisms, such as CBM-Z, RADM2, SAPRC-07 had been successful in studying gas-phase atmospheric chemical processes they all used some lumped organic species to varying degrees. Photochemical Assessment Monitoring Stations (PAMS) has been in use for over ten years and yet it is not clear how the detailed organic species measured by PAMS compare to the lumped model species under regional-scale transport and chemistry interactions. By developing a detailed mechanism specifically for the PAMS organics and embedding this diagnostic model within a regional-scale transport and chemistry model we can then directly compare PAMS observation with regional-scale model simulations. We modify one regional-scale chemical transport model (Taiwan Air Quality Model, TAQM) by adding a submodel with chemical mechanism for interactions of the 56 species observed by PAMS. This submodel then calculates the time evolution of these 56 PAMS species within the environment established by TAQM. It is assumed that TAQM can simulate the overall regional-scale environment including impact of regional-scale transport and time evolution of oxidants and radicals. Therefore we can scale these influences to the PAMS organic species and study their time evolution with their species-specific source functions, meteorological transport, and chemical interactions. Model simulations of each species are compared with PAMS hourly surface measurements. A case study located in a metropolitan area in central Taiwan showed that with wind speeds lower than 3 m/s, when meteorological simulation is comparable with observation, the diurnal pattern of each species performs well with PAMS data. It is found that for many observations meteorological transport is an influence and that local emissions of specific species must be represented correctly. At this time there are still species that cannot be modeled properly. We suspect this is mostly due to lack of information on local

  14. Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor*

    PubMed Central

    Mukund, Susmith; Shang, Yonglei; Clarke, Holly J.; Madjidi, Azadeh; Corn, Jacob E.; Kates, Lance; Kolumam, Ganesh; Chiang, Vicky; Luis, Elizabeth; Murray, Jeremy; Zhang, Yingnan; Hötzel, Isidro; Koth, Christopher M.; Allan, Bernard B.

    2013-01-01

    Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases. PMID:24189067

  15. Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor.

    PubMed

    Mukund, Susmith; Shang, Yonglei; Clarke, Holly J; Madjidi, Azadeh; Corn, Jacob E; Kates, Lance; Kolumam, Ganesh; Chiang, Vicky; Luis, Elizabeth; Murray, Jeremy; Zhang, Yingnan; Hötzel, Isidro; Koth, Christopher M; Allan, Bernard B

    2013-12-13

    Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

  16. Measurement of photosynthesis using PAM technology in a purple sulfur bacterium Thermochromatium tepidum (Chromatiaceae).

    PubMed

    Ritchie, Raymond J; Mekjinda, Nutsara

    2015-01-01

    We demonstrate that Blue-diode-based pulse amplitude modulation (PAM) technology can be used to measure the photosynthetic electron transport rate (ETR) of purple sulfur bacteria (Thermochromatium tepidum, Chromatiaceae). Previous studies showed that PAM technology could be used to estimate photosynthesis in purple nonsulfur bacteria and so PAM technology can be used to estimate photosynthesis of both kinds of purple photosynthetic bacteria. The absorptance of Thermochromatium films on glass fiber disks was measured and used to calculate actual ETR. ETR vs Irradiance (P vs E) curves fitted the waiting-in-line model (ETR = (ETRmax × E/Eopt) × exp (1−E/Eopt)). Yield (Y) was only ≈ 0.3–0.4. Thermochromatium saturates at 325 ± 13.8 μmol photons m(−2) s(−1) or ≈15% sunlight and shows photoinhibition at high irradiances. A pond of Thermochromatium would exhibit classic surface inhibition. Photosynthesis is extremely low in the absence of an electron source: ETR increases in the presence of acetate (5 mol m(−3)) provided as an organic carbon source and also increases in the presence of sulfite (3 mol m(−3)) but not sulfide and is only marginally increased by the presence of Fe(2+). Nonphotochemical quenching does occur in Thermochromatium but at very low levels compared to oxygenic photo-organisms or Rhodopseudomonads.

  17. Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

    PubMed

    Puente-Sánchez, Fernando; Olsson, Sanna; Gómez-Rodriguez, Manuel; Souza-Egipsy, Virginia; Altamirano-Jeschke, Maria; Amils, Ricardo; Parro, Victor; Aguilera, Angeles

    2016-02-01

    The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday.

  18. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

  19. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

    PubMed Central

    2015-01-01

    Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. PMID:26529344

  20. PAMS Photo Image Retrieval Prototype System Design Description

    SciTech Connect

    Conner, M.L., Westinghouse Hanford

    1996-05-02

    This System Design Description (SDD) documents the detail design of the Photo Audio/Visual Management System (PAMS) Photo Image Retrieval Prototype (PPIRP) subsystem. This SDD shows how the software is structured to satisfy the requirements identified in the PAMS Photo Image Prototype Requirements Document. It is a description of the software structure, software components,interfaces, and data that make up the PPIRP subsystem.

  1. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

    PubMed Central

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K.; Zhu, Hu; Chen, Xin; Moy, Sheryl S.; Saddoris, Kara A.; Nikolova, Viktoriya; Farrell, Martilias S.; Wang, Sheng; Mangano, Thomas J.; Deshpande, Deepak A.; Jiang, Alice; Penn, Raymond B.; Jin, Jian; Koller, Beverly H.; Kenakin, Terry; Shoichet, Brian K.; Roth, Bryan L.

    2016-01-01

    At least 120 non-olfactory G protein-coupled receptors in the human genome are ”orphans” for which endogenous ligands are unknown, and many have no selective ligands, hindering elucidation of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Yeast-based screens against GPR68 identified the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. Over 3000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators many of which were confirmed in functional assays. One potent GPR68 modulator—ogerin– suppressed recall in fear conditioning in wild-type, but not in GPR68 knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

  2. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

    PubMed Central

    2015-01-01

    The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase. PMID:26191365

  3. An allosteric model for ribonuclease.

    PubMed Central

    Walker, E J; Ralston, G B; Darvey, I G

    1975-01-01

    Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2':3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve of velocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature. PMID:1167152

  4. Supramolecular Allosteric Cofacial Porphyrin Complexes

    SciTech Connect

    Oliveri, Christopher G.; Gianneschi, Nathan C.; Nguyen, Son Binh T.; Mirkin, Chad A.; Stern, Charlotte L.; Wawrzak, Zdzislaw; Pink, Maren

    2008-04-12

    Nature routinely uses cooperative interactions to regulate cellular activity. For years, chemists have designed synthetic systems that aim toward harnessing the reactivity common to natural biological systems. By learning how to control these interactions in situ, one begins to allow for the preparation of man-made biomimetic systems that can efficiently mimic the interactions found in Nature. To this end, we have designed a synthetic protocol for the preparation of flexible metal-directed supramolecular cofacial porphyrin complexes which are readily obtained in greater than 90% yield through the use of new hemilabile porphyrin ligands with bifunctional ether-phosphine or thioether-phosphine substituents at the 5 and 15 positions on the porphyrin ring. The resulting architectures contain two hemilabile ligand-metal domains (Rh{sup I} or Cu{sup I} sites) and two cofacially aligned porphyrins (Zn{sup II} sites), offering orthogonal functionalities and allowing these multimetallic complexes to exist in two states, 'condensed' or 'open'. Combining the ether-phosphine ligand with the appropriate Rh{sup I} or Cu{sup I} transition-metal precursors results in 'open' macrocyclic products. In contrast, reacting the thioether-phosphine ligand with RhI or CuI precursors yields condensed structures that can be converted into their 'open' macrocyclic forms via introduction of additional ancillary ligands. The change in cavity size that occurs allows these structures to function as allosteric catalysts for the acyl transfer reaction between X-pyridylcarbinol (where X = 2, 3, or 4) and 1-acetylimidazole. For 3- and 4-pyridylcarbinol, the 'open' macrocycle accelerates the acyl transfer reaction more than the condensed analogue and significantly more than the porphyrin monomer. In contrast, an allosteric effect was not observed for 2-pyridylcarbinol, which is expected to be a weaker binder and is unfavorably constrained inside the macrocyclic cavity.

  5. Allosteric cross-talk in chromatin can mediate drug-drug synergy

    PubMed Central

    Adhireksan, Zenita; Palermo, Giulia; Riedel, Tina; Ma, Zhujun; Muhammad, Reyhan; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2017-01-01

    Exploitation of drug–drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug–drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions. PMID:28358030

  6. The concept of allosteric interaction and its consequences for the chemistry of the brain.

    PubMed

    Changeux, Jean-Pierre

    2013-09-20

    Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions.

  7. The Concept of Allosteric Interaction and Its Consequences for the Chemistry of the Brain

    PubMed Central

    Changeux, Jean-Pierre

    2013-01-01

    Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions. PMID:23878193

  8. Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

    SciTech Connect

    Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.; Lovell, Scott; Karanicolas, John

    2012-08-07

    Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

  9. Cristallisation, syncristallisation, et alliages moléculaires entre le lorazépam et l'oxazépam

    NASA Astrophysics Data System (ADS)

    Mascherpa-Corral, D.; Mascherpa, G.; Chauvet, A.

    1993-04-01

    Le polymorphisme et pseudopolymorphisme du lorazépam et oxazépam ont été étudiés par analyse thermique, calorimétrie différentielle à balayage et diffraction de rayons X. Deux formes polymorphes du lorazépam et plusieurs mono et hemisolvates ont été isolés à partir de recristallisation dans divers soivants. Aucun polymorphisme ni solvate n'a été mis en évidence avec l'oxazépam. Les paramètres cristallographiques des phases isolées ont été déterminées. A 160 et 203°C, le lorazépam et l'oxazépam perdent respectivement une molécule d'eau pour donner après réarrangement la quinazolinecarboxaldéhyde correspondante. La syncristallisation des deux benzodiazépines dans le benzène conduit à des alliages moléculaires à miscibilité totale à l'état solide dans tout le domaine de concentration entre l'exazépam el la forme α du lorazépam, elle n'est que partielle avec la forme β. Thermal analysis, differential scanning calorimetry, and X-ray diffraction were carried out to study the polymorphism and pseudopolymorphism of lorazepam and oxazepam. Two polymorphic forms of lorazepam and several mono- or hemisolvates were obtained after recrystallization from various solvents. No polymorphic form or solvate has been found for oxazepam. The crystallographic parameters of these new phases were determined. Lorazepam and oxazepam can lose a molecule of water at 160 and 203°C, respectively, and rearrange to quinazolinecarboxaldehyde. Mixed crystals of the two benzodiazepines, after recrystallization from benzene, lead to molecular alloys with complete solid solubility between oxazepam and the α-form of lorazepam over the whole range of composition but only to partial solubility with the β-form of lorazepam.

  10. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

    PubMed Central

    2015-01-01

    The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure–activity relationships, species differences, “molecular switches”), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013; The 2013 Portoghese Lectureship), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization. PMID:25180768

  11. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: drug discovery targeting allosteric sites.

    PubMed

    Lindsley, Craig W

    2014-09-25

    The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure-activity relationships, species differences, "molecular switches"), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship ( Lindsley , C. W. Adventures in allosteric drug discovery . Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013 ; The 2013 Portoghese Lectureship ), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization.

  12. A comparison of PAMS and air toxics measurements

    NASA Astrophysics Data System (ADS)

    Sistla, Gopal; Aleksic, Nenad

    One of the requirements of the 1990 Clean Air Act Amendments (CAA) is that 1-h ozone nonattainment areas that are classified severe or higher category are required to operate a network of photochemical assessment monitors (PAMS) to provide hourly measurements of volatile organic compounds (VOCs) comprising of Carbon number <12 (C2-C12), along with carbonyl measurements at 3-h intervals during the summer ozone season. Often collocated with PAMS are 24-h-integrated canister and cartridge-based measurements of selected air toxic compounds, thereby providing an opportunity for inter-comparison and validation of both sets of data. In this study, we report such a comparison and estimates of trend for benzene, m-, p- and o-xylene, toluene, ethylbenzene, 1,2,4-trimethylbenzene, formaldehyde and acetaldehyde at Bronx, NY. The analysis shows that hourly PAMS and 24-h-integrated air toxics are in good agreement with each other exhibiting similar trends and that the PAMS with the higher temporal resolution offers information on excursions of the toxic compounds that would be quite useful in assessment of acute health effects. These findings were also found to be applicable to other locations such as South De Kalb, GA; Gary, IN and Lynn, MA.

  13. Iterative nonlinear ISI cancellation in optical tilted filter-based Nyquist 4-PAM system

    NASA Astrophysics Data System (ADS)

    Ju, Cheng; Liu, Na

    2016-09-01

    The conventional double sideband (DSB) modulation and direct detection scheme suffers from severer power fading, linear and nonlinear inter-symbol interference (ISI) caused by fiber dispersion and square-law direct detection. The system's frequency response deteriorates at high frequencies owing to the limited device bandwidth. Moreover, the linear and nonlinear ISI is enhanced induced by the bandwidth limited effect. In this paper, an optical tilted filter is used to mitigate the effect of power fading, and improve the high frequency response of bandwidth limited device in Nyquist 4-ary pulse amplitude modulation (4-PAM) system. Furtherly, iterative technique is introduced to mitigate the nonlinear ISI caused by the combined effects of electrical Nyquist filter, limited device bandwidth, optical tilted filter, dispersion, and square-law photo-detection. Thus, the system's frequency response is greatly improved and the delivery distance can be extended.

  14. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  15. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  16. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  17. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  18. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  19. Transmission of 112(4×28)-Gb/s PAM-4 Signal over 48.6-km SSMF within only 50-GHz Grid

    NASA Astrophysics Data System (ADS)

    Dong, Jian; Hu, Rong

    2016-12-01

    In this paper, a transmission of 112(4×28)-Gb/s PAM-4 signal is experimentally demonstrated within only 50-GHz grid, achieving an optical spectral efficiency (SE) of 2.24 b/s/Hz. For the intensity modulation and direct detection (IM/DD) based PAM-4 transmission, it is the first time to the best of our knowledge that 112-Gb/s PAM-4 signal has been transmitted over 48.6-km standard single mode fiber (SSMF), which is compatible with 50-GHz standard gridding. By employing digital pre-equalization, duobinary encoding/decoding and 7-level based training sequence aided least-mean square (TS-LMS) algorithm, each lane of the 28-Gb/s PAM-4 signal occupies less than 11-GHz optical spectral (3-dB bandwidth), resulting in negligible inter-channel interference for the 4-lane 112-Gb/s PAM-4 signals within 50-GHz grid. The proposed method is bandwidth and computationally efficient, which is thought feasible in the low-cost short reach optical networks.

  20. Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry.

    PubMed

    Peña-Vázquez, E; Pérez-Conde, C; Costas, E; Moreno-Bondi, M C

    2010-08-01

    Test methods are needed to monitor Cu concentrations in reservoirs and water supplies. Dictyosphaerium chlorelloides (Chlorophyta) cells were immobilized in a silicate sol-gel and the toxic effects of Cu(II) were examined using different techniques: fluorescence measurements (using a spectrofluorometer with an optic fiber coupled to a flow cell or a 96-well-plate reader) or by Pulse Amplitude Modulation (PAM) parameters using a portable instrument and the pulse saturation method. Fm' and qN were the most sensitive indicator parameters when performing Cu analysis in water. D. chlorelloides PAM biosensor presented a detection limit of 0.6 mg l(-1) for Cu(II), within the limits to establish if Cu concentrations exceeded regulatory levels. Moreover, a 1.9 mg Cu l(-1) (30 microM) resistant strain of the D. chlorelloides microalgae was produced in order to obtain more selectivity on the metal determination.

  1. Using Profile Analysis via Multidimensional Scaling (PAMS) to Identify Core Profiles from the WMS-III

    ERIC Educational Resources Information Center

    Frisby, Craig L.; Kim, Se-Kang

    2008-01-01

    Profile Analysis via Multidimensional Scaling (PAMS) is a procedure for extracting latent core profiles in a multitest data set. The PAMS procedure offers several advantages compared with other profile analysis procedures. Most notably, PAMS estimates individual profile weights that reflect the degree to which an individual's observed profile…

  2. Field application of PAM as an amendment in deep-tilled US southeastern Coastal Plain soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyacrylamide (PAM) was added to sandy coastal plain soils to improve physical properties and yield. Soils were amended with 8 treatments of linear and cross-linked PAMs or controls. Treatments and controls included the following: 1. spraying a 600 mg/kg solution of linear PAM behind a subsoil shan...

  3. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    PubMed

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

  4. Allosteric substrate switching in a voltage sensing lipid phosphatase

    PubMed Central

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  5. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  6. EFFECTS OF 2-PAM AND EA 1814 ON NEUROMUSCULAR TRANSMISSION. I. EFFECTS OF 2-PAM AND EA 1814 ON THE FROG RECTUS ABDOMINIS MUSCLE PREPARATION

    DTIC Science & Technology

    Contracture of the isolated frog Rectus abdominis muscle was used to study pharmacological properties of 2-PAM (2-pyridine aldoxime methiodide) and...example, concentrations of 2-PAM in excess of 4 x 10 to the -5th power M potentiate contractures of the frog rectus muscle elicited by acetylcholine...2-PAM inhibits the response to the depolarizing agents, decamethonium and carbamylcholine, which are not susceptible to hydrolysis by the ChE of frog

  7. High-throughput screening for GPR119 modulators identifies a novel compound with anti-diabetic efficacy in db/db mice.

    PubMed

    Zhang, Meng; Feng, Yang; Wang, Jia; Zhao, Jianwei; Li, Ting; He, Min; Yang, Dehua; Nosjean, Olivier; Boutin, Jean; Renard, Pierre; Wang, Ming-Wei

    2013-01-01

    G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β cells and enteroendocrine cells. It is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, thereby representing a promising target for the treatment of type 2 diabetes. Although a number of GPR119 agonists were developed, no positive allosteric modulator (PAM) to this receptor has been reported. Here we describe a high-throughput assay for screening GPR119 PAMs and agonists simultaneously. Following screening of a small molecule compound library containing 312,000 synthetic and natural product-derived samples, one potent GPR119 agonist with novel chemical structure, MW1219, was identified. Exposure of MIN6 and GLUTag cells to MW1219 enhanced glucose-stimulated insulin secretion and GLP-1 release; once-daily oral dosing of MW1219 for 6 weeks in diabetic db/db mice reduced hemoglobin A1c (HbA1c) and improved plasma glucose, insulin and GLP-1 levels; it also increased glucose tolerance. The results demonstrate that MW1219 is capable of effectively controlling blood glucose level and may have the potential to be developed as a new class of anti-diabetic agents.

  8. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  9. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.

    PubMed

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-03-03

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.

  10. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  11. Tropical cyclone Pam coastal impact survey in Vanuatu

    NASA Astrophysics Data System (ADS)

    Fritz, H. M.; Pilarczyk, J.; Kosciuch, T. J.; Hong, I.; Rarai, A.; Harrison, M. J.; Jockley, F. R.; Horton, B.

    2015-12-01

    Severe tropical cyclone Pam (Cat. 5, SSHS) crossed the Vanuatu archipelago with sustained winds of 270 km/h on March 13 and 14, 2015 and made landfall on Erromango. Pam caused the worst natural disaster in Vanuatu's recorded history since severe tropical cyclone Uma in 1987. Eleven fatalities were directly attributed to cyclone Pam and mostly due to lack of shelter from airborne debris. On March 6 Pam formed east of the Santa Cruz Islands and intensified while tracking southward along Vanuatu severely affecting the Shefa and Tafea Provinces. An international storm surge reconnaissance team was deployed to Vanuatu from June 3 to 17, 2015 to complement earlier local surveys. Cyclone Pam struck a remote island archipelago particularly vulnerable to the combined cyclonic multi-hazards encompassing extreme wind gusts, massive rainfall and coastal flooding due to a combination of storm surge and storm wave impacts. The team surveyed coastal villages on Epi, the Shepherd Islands (Tongoa and Mataso), Efate (including Lelepa), Erromango, and Tanna. The survey spanned 320 km parallel to the cyclone track between Epi and Tanna encompassing more than 45 sites including the hardest hit settlements. Coastal flooding profiles were surveyed from the shoreline to the limit of inundation. Maximum coastal flood elevations and overland flow depths were measured based on water marks on buildings, scars on trees, rafted debris and corroborated with eyewitness accounts. We surveyed 91 high water marks with characteristic coastal flood levels in the 3 to 7 m range and composed of storm surge with superimposed storm waves. Inundation distances were mostly limited to a few hundred meters. Coral boulders of more than 1 m diameter were measured on Erromango and sediment samples were collected at key sites across the archipelago. Infrastructure damage on traditional and modern structures was assessed. Eyewitnesses were interviewed at most sites to document the chronology of the wind and

  12. Experimental study of PAM-4, CAP-16, and DMT for 100 Gb/s short reach optical transmission systems.

    PubMed

    Zhong, Kangping; Zhou, Xian; Gui, Tao; Tao, Li; Gao, Yuliang; Chen, Wei; Man, Jiangwei; Zeng, Li; Lau, Alan Pak Tao; Lu, Chao

    2015-01-26

    Advanced modulation formats combined with digital signal processing and direct detection is a promising way to realize high capacity, low cost and power efficient short reach optical transmission system. In this paper, we present a detailed investigation on the performance of three advanced modulation formats for 100 Gb/s short reach transmission system. They are PAM-4, CAP-16 and DMT. The detailed digital signal processing required for each modulation format is presented. Comprehensive simulations are carried out to evaluate the performance of each modulation format in terms of received optical power, transmitter bandwidth, relative intensity noise and thermal noise. The performance of each modulation format is also experimentally studied. To the best of our knowledge, we report the first demonstration of a 112 Gb/s transmission over 10km of SSMF employing single band CAP-16 with EML. Finally, a comparison of computational complexity of DSP for the three formats is presented.

  13. Pulse amplitude modulated chlorophyll fluorometer

    DOEpatents

    Greenbaum, Elias; Wu, Jie

    2015-12-29

    Chlorophyll fluorometry may be used for detecting toxins in a sample because of changes in micro algae. A portable lab on a chip ("LOAC") based chlorophyll fluorometer may be used for toxin detection and environmental monitoring. In particular, the system may include a microfluidic pulse amplitude modulated ("PAM") chlorophyll fluorometer. The LOAC PAM chlorophyll fluorometer may analyze microalgae and cyanobacteria that grow naturally in source drinking water.

  14. Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis*♦

    PubMed Central

    Vorontsov, Ivan I.; Wu, Ying; DeLucia, Maria; Minasov, George; Mehrens, Jennifer; Shuvalova, Ludmilla; Anderson, Wayne F.; Ahn, Jinwoo

    2014-01-01

    EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity. PMID:24338016

  15. Novel selective allosteric and bitopic ligands for the S1P(3) receptor.

    PubMed

    Jo, Euijung; Bhhatarai, Barun; Repetto, Emanuela; Guerrero, Miguel; Riley, Sean; Brown, Steven J; Kohno, Yasushi; Roberts, Edward; Schürer, Stephan C; Rosen, Hugh

    2012-12-21

    Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating G protein-coupled receptors for S1P, namely, S1P(1) through S1P(5). Here, we map the S1P(3) binding pocket with a novel allosteric agonist (CYM-5541), an orthosteric agonist (S1P), and a novel bitopic antagonist (SPM-242). With a combination of site-directed mutagenesis, ligand competition assay, and molecular modeling, we concluded that S1P and CYM-5541 occupy different chemical spaces in the ligand binding pocket of S1P(3). CYM-5541 allowed us to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety, and the novel allosteric hydrophobic pocket permits S1P(3) selectivity of CYM-5541 within the highly similar S1P receptor family. However, a novel S1P(3)-selective antagonist, SPM-242, in the S1P(3) pocket occupies the ligand binding spaces of both S1P and CYM-5541, showing its bitopic mode of binding. Therefore, our coordinated approach with biochemical data and molecular modeling, based on our recently published S1P(1) crystal structure data in a highly conserved set of related receptors with a shared ligand, provides a strong basis for the successful optimization of orthosteric, allosteric, and bitopic modulators of S1P(3).

  16. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein.

    PubMed

    Popovych, Nataliya; Tzeng, Shiou-Ru; Tonelli, Marco; Ebright, Richard H; Kalodimos, Charalampos G

    2009-04-28

    The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

  17. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

    SciTech Connect

    Liu, Wei; Chun, Eugene; Thompson, Aaron A.; Chubukov, Pavel; Xu, Fei; Katritch, Vsevolod; Han, Gye Won; Roth, Christopher B.; Heitman, Laura H.; IJzerman, Adriaan P.; Cherezov, Vadim; Stevens, Raymond C.

    2012-08-31

    Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A{sub 2A} adenosine receptor by replacing its third intracellular loop with apocytochrome b{sub 562}RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp{sup 2.50}. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

  18. Full duplex fiber link for alternative wired and wireless access based on SSB optical millimeter-wave with 4-PAM signal

    NASA Astrophysics Data System (ADS)

    Ma, Jianxin; Zhang, Junjie

    2015-03-01

    A novel full-duplex fiber-wireless link based on single sideband (SSB) optical millimeter (mm)-wave with 10 Gbit/s 4-pulse amplitude modulation (PAM) signal is proposed to provide alternative wired and 40 GHz wireless accesses for the user terminals. The SSB optical mm-wave with 4-PAM signal consists of two tones: one bears the 4-PAM signal and the other is unmodulated with high power. After transmission over the fiber to the hybrid optical network unit (HONU), the SSB optical mm-wave signal can be decomposed by fiber Bragg gratings (FBGs) as the SSB optical mm-wave signal with reduced carrier-to-sideband ratio (the baseband 4-PAM optical signal) and the uplink optical carrier for the wireless (wired) access. This makes the HONU free from the laser source. For the uplink, since the wireless access signal is converted to the baseband by power detection, both the transmitter in the HONU and the receiver in optical line terminal (OLT) are co-shared for both wireless and wired accesses, which makes the full duplex link much simpler. In our scheme, the optical electrical field of the square-root increment level 4-PAM signal assures an equal level spacing receiving for both the downlink wired and wireless accesses. Since the downlink wireless signal is down-converted to the baseband by power detection, RF local oscillator is unnecessary. To confirm the feasibility of our proposed scheme, a simulation full duplex link with 40 GHz SSB optical mm-wave with 10 Gbit/s 4-PAM signal is built. The simulation results show that both down- and up-links for either wired or wireless access can keep good performance even if the link length of the SSMF is extended to 40 km.

  19. Regulation by membrane fluidity of the allosteric behavior of the (Ca2)-adenosine triphosphatase from Escherichia coli.

    PubMed

    Siñeriz, F; Bloj, B; Farías, R N; Trucco, R E

    1973-09-01

    The allosteric properties of the membrane-bound (Ca(2+))-adenosine triphosphatase of an unsaturated fatty acid auxotroph of Escherichia coli were studied in membranes with different fatty acid compositions. The Hill coefficient of the inhibition by Na(+) ranged from 1.4, in the case where the auxotroph was grown with cis-vaccenic acid as supplement, to 2.8 when grown on linolenic acid. The results indicate that no fatty acid is particularly involved in the allosteric phenomena. A correlation between the values of the Hill coefficient and the double bond index or the ratio of the double bond index saturated to the fatty acids of the membrane was found. These facts are interpreted as a modulation by the membrane fluidity of the allosteric behavior of the membrane-bound enzyme. The general biological character of this phenomenon is discussed in this paper.

  20. Analysis of the Performance of a PAM/PPM/OOK System Operating with OCDMA, under Nonlinear Optical Effects in Optical Fiber Propagation

    NASA Astrophysics Data System (ADS)

    Correia, D. G.; Sales, J. C.; Pinto, P. V. F.; Moura, L. P.; Ferreira, A. C.; Menezes, J. W. M.; Guimarães, G. F.; Sombra, A. S. B.

    2016-06-01

    In this article, we present a numerical simulation study of encoding, decoding and propagation performance of short optical pulses and words with modulations OOK, PAM and PPM in OCDMA systems (Optical Code Division Multiple Access). The encoding and decoding of short pulses are obtained through fiber Bragg grating(FBG - FBG optical) devices, where the codes are inserted through discrete jumps in the optical phase (±π) where Gold codes were used. A figure of merit (SNR - Signal to Noise Ratio) was obtained to quantify the interference in propagation of short optical pulses. An increase in the temporal width was observed. For decoded pulses due to the nonlinearity effect, we observed an increase of 1.3 ps considering the propagation with γ=3 W-1 km-1 and γ=24 W-1 km-1. Analysis of coding and decoding words "a" and "w" was done. Considering the propagation (with γ=9 W-1 km-1) of a word "w", an error occurred in all modulations except for simultaneous PPM/PAM modulation, which is associated to the better autocorrelation characteristics obtained with the OOK, PAM and PPM modulations alone, and could double the transmission rate. The nonlinear effects directly affect the process of the autocorrelation codes due to interference from adjacent chip components of the code.

  1. Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2014-01-01

    A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This

  2. An engineered chorismate mutase with allosteric regulation.

    PubMed

    Zhang, Sheng; Wilson, David B; Ganem, Bruce

    2003-07-17

    Besides playing a central role in phenylalanine biosynthesis, the bifunctional P-protein in Eschericia coli provides a unique model system for investigating whether allosteric effects can be engineered into protein catalysts using modular regulatory elements. Previous studies have established that the P-protein contains three distinct domains whose functions are preserved, even when separated: chorismate mutase (residues 1-109), prephenate dehydratase (residues 101-285), and an allosteric domain (residues 286-386) for feedback inhibition by phenylalanine. By deleting the prephenate dehydrase domain, a functional chorismate mutase linked directly to the phenylalanine binding domain has been engineered and overexpressed. This manuscript reports the catalytic properties of the mutase in the absence and presence of phenylalanine.

  3. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  4. Using Profile Analysis via Multidimensional Scaling (PAMS) to identify core profiles from the WMS-III.

    PubMed

    Frisby, Craig L; Kim, Se-Kang

    2008-03-01

    Profile Analysis via Multidimensional Scaling (PAMS) is a procedure for extracting latent core profiles in a multitest data set. The PAMS procedure offers several advantages compared with other profile analysis procedures. Most notably, PAMS estimates individual profile weights that reflect the degree to which an individual's observed profile approximates the shape and scatter of latent core profiles. The PAMS procedure was applied to index scores of nonreplicated participants from the standardization sample (N = 1,033) for the Wechsler Memory Scale--Third Edition (D. Tulsky, J. Zhu, & M. F. Ledbetter, 2002). PAMS extracted discrepant visual memory and auditory memory versus working memory core profiles for the complete 16- to 89-year-old sample and discrepant working memory and auditory memory versus working memory core profiles for the 75- to 89-year-old cohort. Implications for use of PAMS in future research are discussed.

  5. Recommendations for Review of TRADOC Pam 351-4(T).

    DTIC Science & Technology

    1981-07-08

    CHAPTER 8 TRADOC PAM 351-4(T) A generic procedure must be qualified with statement like: Do x except in cases of... Do x only when circumstance y...may also not be practical or necessary in every case . 59 Neeker: It should be DTD for the endorsement of JTA plans. BOWLES: The big problem is what is...Task analsls is different when you deal with new equipment or procedures. Menchaca: In some cases of this type I have used vendors’ manuals. 90 If

  6. Single-lane 180  Gb/s DB-PAM-4-signal transmission over an 80  km DCF-free SSMF link.

    PubMed

    Zhang, Qiang; Stojanovic, Nebojsa; Wei, Jinlong; Xie, Changsong

    2017-02-15

    We experimentally demonstrate the generation and successful transmission of a single-lane 180 Gb/s (90 GBd) duo-binary four-level pulse-amplitude modulation (DB-PAM-4) signal over an 80 km standard single-mode fiber with a net data-rate of 150 Gb/s, enabled by a dual-drive Mach-Zehnder modulator-aided dispersion pre-compensation. A net data rate of 168 Gb/s is also achievable, for ranges up to 40 km. To mitigate bandwidth limitations and error spreading, pre-coded DB-PAM-4 is used; the pre-coding provides a gain of approximately 1 dB. A Volterra filter and a maximum likelihood sequence estimator are used at the receiver side to reduce the linear and nonlinear distortions.

  7. Allosteric ACTion: the varied ACT domains regulating enzymes of amino-acid metabolism.

    PubMed

    Lang, Eric J M; Cross, Penelope J; Mittelstädt, Gerd; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a βαββαβ topology leading to a four-stranded β4β1β3β2 antiparallel sheet with two antiparallel helices on one face. Extensive sequence variation requires a combined sequence/structure/function analysis for identification of the ACT domain. Common features include highly varied modes of self-association of ACT domains, ligand binding at domain interfaces, and transmittal of allosteric signals through conformational changes and/or the manipulation of quaternary equilibria. A recent example illustrates the relatively facile adoption of this versatile module of allostery by gene fusion.

  8. Positive modulators of the α7 nicotinic receptor against neuroinflammation and cognitive impairment in Alzheimer's disease.

    PubMed

    Echeverria, Valentina; Yarkov, Alex; Aliev, Gjumrakch

    2016-09-01

    Evidence so far indicates that therapies targeting a single aspect of Alzheimer's disease (AD) have no sufficient efficacy in diminishing long-term the progression of AD. Neuroinflammation is an early event during the development of the disease and it is thought to exacerbate the abnormal aggregation of the amyloid beta peptide (Aβ) and the microtubule associated protein Tau. Inhibition of gliosis is considered fundamental to reduce neuroinflammation, oxidative stress, apoptosis and synaptic dysfunction driving the progression of AD. Drugs that are able to target more than one aspect of the pathology may have higher chances of success. Modulators of α7 nicotinic acetylcholine receptors (α7nAChRs) such as nicotine and some of its derivatives have this potential because of their anti-inflammatory, anti-apoptotic, pro-cognitive and anti-protein aggregation effects. However, the rapid desensitization of α7nAChRs is considered an important factor limiting its potential therapeutic use. In here, in light of current evidence, the objective of this review is to discuss the advantages and potential therapeutic value of positive allosteric modulators (PAMs) of the nAChRs in halting or delaying the progression of AD by diminishing neuroinflammation, abnormal protein aggregation and synaptic dysfunction.

  9. PAM and Copper – a Gene/Nutrient Interaction Critical to Nervous System Function

    PubMed Central

    Bousquet-Moore, Danielle; Mains, Richard E.; Eipper, Betty A.

    2013-01-01

    Peptidylgycine α-amidating monooxygenase (PAM), a highly conserved copper-dependent enzyme, is essential for the synthesis of all amidated neuropeptides. Biophysical studies revealed that the binding of copper to PAM affects its structure, and cell biological studies demonstrated that the endocytic trafficking of PAM was sensitive to copper. We review data indicating that genetic reduction of PAM expression and mild copper deficiency in mice cause similar alterations in several physiological functions known to be regulated by neuropeptides - thermal regulation, seizure sensitivity and anxiety-like behavior. PMID:20648645

  10. Nanoprecipitated catestatin released from pharmacologically active microcarriers (PAMs) exerts pro-survival effects on MSC.

    PubMed

    Angotti, C; Venier-Julienne, M C; Penna, C; Femminò, S; Sindji, L; Paniagua, C; Montero-Menei, C N; Pagliaro, P

    2016-11-22

    Catestatin (CST), a fragment of Chromogranin-A, exerts angiogenic, arteriogenic, vasculogenic and cardioprotective effects. CST is a very promising agent for revascularization purposes, in "NOOPTION" patients. However, peptides have a very short half-life after administration and must be conveniently protected. Fibronectin-coated pharmacologically active microcarriers (FN-PAM), are biodegradable and biocompatible polymeric microspheres that can convey mesenchymal stem cell (MSCs) and therapeutic proteins delivered in a prolonged manner. In this study, we first evaluated whether a small peptide such as CST could be nanoprecipitated and incorporated within FN-PAMs. Subsequently, whether CST may be released in a prolonged manner by functionalized FN-PAMs (FN-PAM-CST). Finally, we assessed the effect of CST released by FN-PAM-CST on the survival of MSCs under stress conditions of hypoxia-reoxygenation. An experimental design, modifying three key parameters (ionic strength, mixing and centrifugation time) of protein nanoprecipitation, was used to define the optimum condition for CST. An optimal nanoprecipitation yield of 76% was obtained allowing encapsulation of solid CST within FN-PAM-CST, which released CST in a prolonged manner. In vitro, MSCs adhered to FN-PAMs, and the controlled release of CST from FN-PAM-CST greatly limited hypoxic MSC-death and enhanced MSC-survival in post-hypoxic environment. These results suggest that FN-PAM-CST are promising tools for cell-therapy.

  11. Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

    PubMed

    Ahn, Seungkirl; Kahsai, Alem W; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L; Strachan, Ryan T; Staus, Dean P; Wingler, Laura M; Sun, Lillian D; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T; Hansen, Gwenn M; Burnett, Michael B; Lamerdin, Jane E; Bassoni, Daniel L; Gavino, Bryant J; Husemoen, Gitte; Olsen, Eva K; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J

    2017-02-14

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

  12. Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

    PubMed Central

    Ahn, Seungkirl; Kahsai, Alem W.; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L.; Strachan, Ryan T.; Staus, Dean P.; Wingler, Laura M.; Sun, Lillian D.; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T.; Hansen, Gwenn M.; Burnett, Michael B.; Lamerdin, Jane E.; Bassoni, Daniel L.; Gavino, Bryant J.; Husemoen, Gitte; Olsen, Eva K.; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J.

    2017-01-01

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects. PMID:28130548

  13. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  14. Understanding the Critical Parameters of the PAMS Mandrel Fabrication Process

    DOE PAGES

    Bhandarkar, Suhas; Paguio, Reny; Elsner, Fred; ...

    2016-07-05

    As a part of an effort to continually better the roundness and roughness of ablator capsules, we looked at improving the same for the poly(alphamethylstyrene) or PAMS mandrels used to make the plastic capsules. The importance of this work is based on the fact that the surface properties of the mandrels set the lower limit for the ultimate attributes of the ablator capsule. These mandrels are made using an elegant double-emulsion process that uses the isotropic forces brought about by hydrostatic pressure and interfacial tension to seek sphericity. This paper describes the reasoning that led to investigating the so-called curingmore » process where a solid PAMS shell is generated from a solution phase for achieving this goal. Using modeling to account for the mass transfer of the fluorobenzene solvent phase, we demonstrate that it is the control of the conditions through the percolation point of the system that leads to better mandrels. These concepts were implemented into the fabrication process to demonstrate significant improvements of the roundness of the mandrels.« less

  15. Understanding the Critical Parameters of the PAMS Mandrel Fabrication Process

    SciTech Connect

    Bhandarkar, Suhas; Paguio, Reny; Elsner, Fred; Hoover, Denise

    2016-07-05

    As a part of an effort to continually better the roundness and roughness of ablator capsules, we looked at improving the same for the poly(alphamethylstyrene) or PAMS mandrels used to make the plastic capsules. The importance of this work is based on the fact that the surface properties of the mandrels set the lower limit for the ultimate attributes of the ablator capsule. These mandrels are made using an elegant double-emulsion process that uses the isotropic forces brought about by hydrostatic pressure and interfacial tension to seek sphericity. This paper describes the reasoning that led to investigating the so-called curing process where a solid PAMS shell is generated from a solution phase for achieving this goal. Using modeling to account for the mass transfer of the fluorobenzene solvent phase, we demonstrate that it is the control of the conditions through the percolation point of the system that leads to better mandrels. These concepts were implemented into the fabrication process to demonstrate significant improvements of the roundness of the mandrels.

  16. Extending the Technology Acceptance Model: Policy Acceptance Model (PAM)

    NASA Astrophysics Data System (ADS)

    Pierce, Tamra

    There has been extensive research on how new ideas and technologies are accepted in society. This has resulted in the creation of many models that are used to discover and assess the contributing factors. The Technology Acceptance Model (TAM) is one that is a widely accepted model. This model examines people's acceptance of new technologies based on variables that directly correlate to how the end user views the product. This paper introduces the Policy Acceptance Model (PAM), an expansion of TAM, which is designed for the analysis and evaluation of acceptance of new policy implementation. PAM includes the traditional constructs of TAM and adds the variables of age, ethnicity, and family. The model is demonstrated using a survey of people's attitude toward the upcoming healthcare reform in the United States (US) from 72 survey respondents. The aim is that the theory behind this model can be used as a framework that will be applicable to studies looking at the introduction of any new or modified policies.

  17. The GABAB receptor positive modulator BHF177 attenuated anxiety, but not conditioned fear, in rats

    PubMed Central

    Li, Xia; Kaczanowska, Katarzyna; Finn, M. G.; Markou, Athina; Risbrough, Victoria B.

    2015-01-01

    GABAB (γ-aminobutyric acid B) receptors may be a therapeutic target for anxiety disorders. Here we characterized the effects of the GABAB receptor positive allosteric modulator (PAM) BHF177 on conditioned and unconditioned physiological responses to threat in the light-enhanced startle (LES), stress-induced hyperthermia, and fear-potentiated startle (FPS) procedures in rats. The effects of BHF177 on LES were compared with those of the GABAB receptor agonists baclofen and CGP44532, and the positive control buspirone, a 5-HT1A receptor partial agonist with anxiolytic activity in humans. Baclofen (0.4, 0.9 and 1.25 mg/kg) and CGP44532 (0.065, 0.125 and 0.25 mg/kg) administration had significant sedative, but not anxiolytic, activity reflected in overall decrease in the startle response in the LES tests. BHF177 (10, 20 and 40 mg/kg) had no effect on LES, nor did it produce an overall sedative effect. Interesting, however, when rats were grouped by high and low LES responses, BHF177 had anxiolytic-like effects only on LES in high, but not low, LES responding rats. BHF177 also blocked stress-induced hyperthermia, but had no effect on conditioned fear responses in the FPS test. Buspirone (1 and 3 mg/kg) had an anxiolytic-like profile in both LES and FPS tests. These results indicate that BHF177 may specifically attenuate unconditioned anxiety in individuals that exhibit a high anxiety state, and has fewer sedative effects than direct agonists. Thus, BHF177 or other GABAB receptor PAMs may be promising compounds for alleviating increased anxiety seen in various psychiatric disorders with a superior side-effect profile compared to GABAB receptor agonists. PMID:26002628

  18. Mimicking the Regulation Step of Fe-Monooxygenases: Allosteric Modulation of Fe(IV) -Oxo Formation by Guest Binding in a Dinuclear Zn(II) -Fe(II) Calix[6]arene-Based Funnel Complex.

    PubMed

    Ségaud, Nathalie; De Thomasson, Constance; Daverat, Caroline; Sénéchal-David, Katell; Dos Santos, Amandine; Steinmetz, Vincent; Maître, Philippe; Rebilly, Jean-Noël; Banse, Frédéric; Reinaud, Olivia

    2017-02-24

    A heteroditopic ligand associated with a calix[6]arene scaffold bearing a tris(imidazole) coordinating site at its small rim and an amine/pyridine ligand at its large rim has been prepared, and its regioselective coordination to Zn(II) at the small rim and Fe(II) in the amine/pyridine ligand has been achieved. The heterodinuclear complex obtained displays an overall cone conformation capped by the tris(imidazole)Zn(II) moiety and bears a non-heme Fe(II) complex at its base. Each of the metal centers exhibits one labile position, allowing the coordination inside the cavity of a guest alkylamine at Zn(II) and the generation of reaction intermediates (Fe(III) (OOH) and Fe(IV) O) at the large rim. A dependence between the chain length of the encapsulated alkylamine and the distribution of Fe(III) (OOH) intermediates and Fe(III) (OMe) is observed. In addition, it is shown that the generation of the Fe(IV) O intermediate is enhanced by addition of the alkylamine guest. Hence, this supramolecular system gathers the three levels of reactivity control encountered in oxidoreductases: i) control of the Fe(II) redox properties through its first coordination sphere, allowing us to generate high valent reactive species; ii) control of guest binding through a hydrophobic funnel that drives its alkyl chain next to the reactive iron complex, thus mimicking the binding pocket of natural systems; iii) guest-modulated reactivity of the Fe(II) center towards oxidants.

  19. Infiltration and Erosion in Soils Treated with Dry PAM of Two Molecular Weights and Phosphogypsum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil surface application of dissolved linear polyacrylamide (PAM) of high molecular weight (MW) can mitigate seal formation, runoff and erosion, especially when added with a source of electrolytes (e.g., gypsum). Practical difficulties associated with PAM solution application prohibited commercial u...

  20. PHBV/PAM scaffolds with local oriented structure through UV polymerization for tissue engineering.

    PubMed

    Ke, Yu; Wu, Gang; Wang, Yingjun

    2014-01-01

    Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100  μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

  1. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all components of the SIP, Virginia must implement the program as submitted and approved by EPA....

  2. 40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Delaware SIP. As with all components of the SIP, Delaware must implement the program as submitted and approved by EPA....

  3. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all components of the SIP, Virginia must implement the program as submitted and approved by EPA....

  4. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all components of the SIP, Virginia must implement the program as submitted and approved by EPA....

  5. 40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Delaware SIP. As with all components of the SIP, Delaware must implement the program as submitted and approved by EPA....

  6. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all components of the SIP, Virginia must implement the program as submitted and approved by EPA....

  7. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all components of the SIP, Virginia must implement the program as submitted and approved by EPA....

  8. 40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Delaware SIP. As with all components of the SIP, Delaware must implement the program as submitted and approved by EPA....

  9. 40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Delaware SIP. As with all components of the SIP, Delaware must implement the program as submitted and approved by EPA....

  10. 40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as... Stations (PAMS) Program on September 11, 1995 and made it part of the Delaware SIP. As with all components of the SIP, Delaware must implement the program as submitted and approved by EPA....

  11. BLOOD PLASMA LEVELS AND ELIMINATION OF SALTS OF 2-PAM IN MAN AFTER ORAL ADMINISTRATION,

    DTIC Science & Technology

    salts of 2-PAM in varying amounts. A measurable amount of oxime was found in blood plasma within about 15 minutes; the concentration rose rapidly...level of oxime 3.5 fold. The biological half-life in man of the 2-PAM salts given orally, calculated from blood plasma values and urinary excretion rates

  12. Allosteric models for multimeric proteins: oxygen-linked effector binding in hemocyanin.

    PubMed

    Menze, Michael A; Hellmann, Nadja; Decker, Heinz; Grieshaber, Manfred K

    2005-08-02

    In many crustaceans, changing concentrations of several low molecular weight compounds modulates hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the pigment. The nonphysiological effector caffeine and the physiological modulator urate, the latter accumulating in the hemolymph of the lobster Homarus vulgaris during hypoxia, increase hemocyanin oxygen affinity and decrease cooperativity of oxygen binding. To derive a model that describes the mechanism of allosteric interaction between hemocyanin and oxygen in the presence of urate or caffeine, studies of oxygen, urate, and caffeine binding to hemocyanin were performed. Exposure of lobster hemocyanin to various pH values between 7.25 and 8.15 resulted in a decrease of p50. In this pH interval, p50 decreases from 95 to 11 Torr without effectors and from 49 to 6 Torr and from 34 to 5 Torr in the presence of 1 mM urate or caffeine, respectively. Thus, the allosteric effects induced by protons and urate or caffeine are coupled. In contrast, isothermal titration calorimetry did not reveal any differences in binding enthalpy (DeltaH degrees ) for urate or caffeine under either normoxic or hypoxic conditions at different pH values. Despite these apparently conflicting results, they can be explained by the nested MWC model if two different types of modulator binding sites are assumed, an allosteric and a nonallosteric type of site. Simulations of in vivo conditions with this model indicate that the naturally occurring modulator urate is physiologically relevant in H. vulgaris only during hypoxic conditions, i.e., either during environmental oxygen limitation or extensive exercise.

  13. Identification of Allosteric Disulfides from Prestress Analysis

    PubMed Central

    Zhou, Beifei; Baldus, Ilona B.; Li, Wenjin; Edwards, Scott A.; Gräter, Frauke

    2014-01-01

    Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states. PMID:25099806

  14. Identification of an allosteric binding site for RORγt inhibition

    PubMed Central

    Scheepstra, Marcel; Leysen, Seppe; van Almen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

  15. Targeting allosteric disulphide bonds in cancer.

    PubMed

    Hogg, Philip J

    2013-06-01

    Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.

  16. Multi-professional staff development programme for PAMs.

    PubMed

    Wray, N

    2001-01-01

    A unique, accredited, multi-professional education programme for junior staff in professions allied to medicine (PAMs) developed by Durham and Teesside Education and Training Consortium in collaboration with local trusts and social services is described. Designed to ensure competence to practice in line with clinical governance, it aims to strengthen multi-professional working practice through a greater understanding of roles and of the way the national health service (NHS) works. It enables staff to analyse their own learning needs and to employ self-directed learning skills for life-long learning. Using problem-based learning, it uses real clinical cases as triggers to promote critical evaluation and reasoning and the development of multi-professional care pathways.

  17. Exploring with PAM: Prospecting ANTS Missions for Solar System Surveys

    NASA Technical Reports Server (NTRS)

    Clark, P. E.; Rilee, M. L.; Curtis, S. A.

    2003-01-01

    ANTS (Autonomous Nano-Technology Swarm), a large (1000 member) swarm of nano to picoclass (10 to 1 kg) totally autonomous spacecraft, are being developed as a NASA advanced mission concept. ANTS, based on a hierarchical insect social order, use an evolvable, self-similar, hierarchical neural system in which individual spacecraft represent the highest level nodes. ANTS uses swarm intelligence attained through collective, cooperative interactions of the nodes at all levels of the system. At the highest levels this can take the form of cooperative, collective behavior among the individual spacecraft in a very large constellation. The ANTS neural architecture is designed for totally autonomous operation of complex systems including spacecraft constellations. The ANTS (Autonomous Nano Technology Swarm) concept has a number of possible applications. A version of ANTS designed for surveying and determining the resource potential of the asteroid belt, called PAM (Prospecting ANTS Mission), is examined here.

  18. Salvinorin A: allosteric interactions at the mu-opioid receptor.

    PubMed

    Rothman, Richard B; Murphy, Daniel L; Xu, Heng; Godin, Jonathan A; Dersch, Christina M; Partilla, John S; Tidgewell, Kevin; Schmidt, Matthew; Prisinzano, Thomas E

    2007-02-01

    Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.

  19. ANTS/PAM: Future Exploration of the Asteroid Belt

    NASA Astrophysics Data System (ADS)

    Clark, P. E.; Curtis, S. A.; Rilee, M. L.; Cheung, C. Y.

    2004-05-01

    The Autonomous Nano-Technology Swarm (ANTS) is applied to the Prospecting Asteroid Mission (PAM) concept, as part of a NASA RASC study. The ANTS architecture is inspired by success of social insect colonies, based on the division of labor within the colonies: 1) within their specialties, individual specialists generally outperform general-ists, and 2) with sufficiently efficient social interaction and coordination, the group of specialists generally outper-forms the group of generalists. ANTS as applied to PAM involves a thousand individual specialist `sciencecraft', one subswarm per target, in an environment where detection and tracking of irregular, infrequent targets is a major chal-lenge. Workers, carry and operate eight to nine different scientific instruments, including spectrometers, ranging and radio science devices, imagers. The remaining specialists, Messenger/Rulers, provide communication and coordina-tion. The non-expendable propulsion system is based on autonomously deployable and configurable solar sails, a system suitable to a low gravity environment. The design of the neural basis function requires a minimum of 4 or 5 specialists for collective decision making. Allowing for ten instrument specialist teams and compensating for antici-pated high attrition, we calculate an initial minimum of 100 per subswarm should allow characterization of hundreds of asteroids. The difficulty in observing irregular, rapidly moving, poorly illuminated objects is largely overcome by the ANT sciencecraft capability to optimize conditions for each instrument. Components are composed of carbon nanotubules reversibly deployable from NEMS nodes, allowing 100 times decrease in packaging volume. 1000 smart 10 centimeter, 1 kg cubic boxes create a 1000 kg 1 meter cube.

  20. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

    PubMed

    Young, Gareth T; Zwart, Ruud; Walker, Alison S; Sher, Emanuele; Millar, Neil S

    2008-09-23

    Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

  1. Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

    PubMed

    Barman, Arghya; Hamelberg, Donald

    2016-08-25

    Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's. Experimental studies suggest that the catalytic domain and the noncatalytic WW domain are allosterically coupled; however, an atomic level description of the dynamics associated with the interdomain communication is lacking. We show that binding of the substrate to the WW domain is directly coupled to the dynamics of the catalytic domain, causing rearrangement of the residue-residue contact dynamics from the WW domain to the catalytic domain. The binding affinity of the substrate in the catalytic domain is also enhanced upon binding of the substrate to the WW domain. Modulation of the dynamics of the catalytic domain upon binding of the substrate to the WW domain leads to prepayment of the entropic cost of binding the substrate to the catalytic domain. This study shows that Ile 28 at the interfacial region between the catalytic and WW domains is certainly one of the residues responsible for bridging the communication between the two domains. The results complement previous experiments and provide valuable atomistic insights into the role of dynamics and possible entropic contribution to the allosteric mechanism of proteins.

  2. Emerging Computational Methods for the Rational Discovery of Allosteric Drugs

    PubMed Central

    2016-01-01

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages. PMID:27074285

  3. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  4. Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

    PubMed Central

    Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

    2013-01-01

    Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

  5. Pam heterozygous mice reveal essential role for Cu in amygdalar behavioral and synaptic function

    PubMed Central

    Gaier, Eric D; Eipper, Betty A; Mains, Richard E

    2014-01-01

    Cu is an essential element with many biological roles, but its roles in the mammalian nervous system are poorly understood. Mice deficient in the cuproenzyme peptidylglycine α-amidating monooxygenase (PAM+/− mice) were initially generated to study neuropeptide amidation. PAM+/− mice exhibited profound deficits in a few behavioral tasks, including enhancements in innate fear along with deficits in acquired fear. Interestingly, several PAM+/− phenotypes were recapitulated in Cu restricted wildtype mice and rescued in Cu supplemented PAM+/− mice. These behaviors correspond to enhanced excitability and deficient synaptic plasticity in the amygdala of PAM+/− mice, which are also rescued by Cu supplementation. Cu and ATP7A