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Sample records for allosteric modulators pams

  1. 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

  2. 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 Central

    2015-01-01

    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. PMID:24735492

  3. 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. PMID:24735492

  4. 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

  5. Allosteric modulation of caspases.

    PubMed

    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  6. Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor.

    PubMed

    Burford, Neil T; Clark, Mary J; Wehrman, Tom S; Gerritz, Samuel W; Banks, Martyn; O'Connell, Jonathan; Traynor, John R; Alt, Andrew

    2013-06-25

    μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin-recruitment assay. PMID:23754417

  7. 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

  8. Structurally Similar Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors Exhibit Five Distinct Pharmacological Effects*

    PubMed Central

    Gill-Thind, JasKiran K.; Dhankher, Persis; D'Oyley, Jarryl M.; Sheppard, Tom D.; Millar, Neil S.

    2015-01-01

    Activation of nicotinic acetylcholine receptors (nAChRs) is associated with the binding of agonists such as acetylcholine to an extracellular site that is located at the interface between two adjacent receptor subunits. More recently, there has been considerable interest in compounds, such as positive and negative allosteric modulators (PAMs and NAMs), that are able to modulate nAChR function by binding to distinct allosteric sites. Here we examined a series of compounds differing only in methyl substitution of a single aromatic ring. This series of compounds includes a previously described α7-selective allosteric agonist, cis-cis-4-p-tolyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4MP-TQS), together with all other possible combinations of methyl substitution at a phenyl ring (18 additional compounds). Studies conducted with this series of compounds have revealed five distinct pharmacological effects on α7 nAChRs. These five effects can be summarized as: 1) nondesensitizing activation (allosteric agonists), 2) potentiation associated with minimal effects on receptor desensitization (type I PAMs), 3) potentiation associated with reduced desensitization (type II PAMs), 4) noncompetitive antagonism (NAMs), and 5) compounds that have no effect on orthosteric agonist responses but block allosteric modulation (silent allosteric modulators (SAMs)). Several lines of experimental evidence are consistent with all of these compounds acting at a common, transmembrane allosteric site. Notably, all of these chemically similar compounds that have been classified as nondesensitizing allosteric agonists or as nondesensitizing (type II) PAMs are cis-cis-diastereoisomers, whereas all of the NAMs, SAMs, and type I PAMs are cis-trans-diastereoisomers. Our data illustrate the remarkable pharmacological diversity of allosteric modulators acting on nAChRs. PMID:25516597

  9. Are AMPA receptor positive allosteric modulators potential pharmacotherapeutics for addiction?

    PubMed

    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

  10. 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

  11. Allosteric modulation of glycine receptors

    PubMed Central

    Yevenes, Gonzalo E; Zeilhofer, Hanns Ulrich

    2011-01-01

    Inhibitory (or strychnine sensitive) glycine receptors (GlyRs) are anion-selective transmitter-gated ion channels of the cys-loop superfamily, which includes among others also the inhibitory γ-aminobutyric acid receptors (GABAA receptors). While GABA mediates fast inhibitory neurotransmission throughout the CNS, the action of glycine as a fast inhibitory neurotransmitter is more restricted. This probably explains why GABAA receptors constitute a group of extremely successful drug targets in the treatment of a wide variety of CNS diseases, including anxiety, sleep disorders and epilepsy, while drugs specifically targeting GlyRs are virtually lacking. However, the spatially more restricted distribution of glycinergic inhibition may be advantageous in situations when a more localized enhancement of inhibition is sought. Inhibitory GlyRs are particularly relevant for the control of excitability in the mammalian spinal cord, brain stem and a few selected brain areas, such as the cerebellum and the retina. At these sites, GlyRs regulate important physiological functions, including respiratory rhythms, motor control, muscle tone and sensory as well as pain processing. In the hippocampus, RNA-edited high affinity extrasynaptic GlyRs may contribute to the pathology of temporal lobe epilepsy. Although specific modulators have not yet been identified, GlyRs still possess sites for allosteric modulation by a number of structurally diverse molecules, including alcohols, neurosteroids, cannabinoids, tropeines, general anaesthetics, certain neurotransmitters and cations. This review summarizes the present knowledge about this modulation and the molecular bases of the interactions involved. PMID:21557733

  12. Molecular determinants of positive allosteric modulation of the human metabotropic glutamate receptor 2

    PubMed Central

    Farinha, A; Lavreysen, H; Peeters, L; Russo, B; Masure, S; Trabanco, A A; Cid, J; Tresadern, G

    2015-01-01

    Background and Purpose The activation of the metabotropic glutamate receptor 2 (mGlu2) reduces glutamatergic transmission in brain regions where excess excitatory signalling is implicated in disorders such as anxiety and schizophrenia. Positive allosteric modulators (PAMs) can provide a fine-tuned potentiation of these receptors' function and are being investigated as a novel therapeutic approach. An extensive set of mutant human mGlu2 receptors were used to investigate the molecular determinants that are important for positive allosteric modulation at this receptor. Experimental Approach Site-directed mutagenesis, binding and functional assays were employed to identify amino acids important for the activity of nine PAMs. The data from the radioligand binding and mutagenesis studies were used with computational docking to predict a binding mode at an mGlu2 receptor model based on the recent structure of the mGlu1 receptor. Key Results New amino acids in TM3 (R635, L639, F643), TM5 (L732) and TM6 (W773, F776) were identified for the first time as playing an important role in the activity of mGlu2 PAMs. Conclusions and Implications This extensive study furthers our understanding of positive allosteric modulation of the mGlu2 receptor and can contribute to improved future design of mGlu2 PAMs. PMID:25571949

  13. Allosteric modulators of the extracellular calcium receptor.

    PubMed

    Nemeth, E F

    2013-01-01

    The extracellular calcium receptor (CaR) is a Family C G protein-coupled receptor that controls systemic Ca2+ homeostasis, largely by regulating the secretion of parathyroid hormone (PTH). Ligands that activate the CaR have been termed calcimimetics and are classified as either Type I (agonists) or Type II (allosteric activators) and effectively inhibit the secretion of PTH. CaR antagonists have been termed calcilytics and all act allosterically to stimulate secretion of PTH. The calcimimetic cinacalcet has been approved for treating parathyroid cancer and secondary hyperparathyroidism in patients on renal replacement therapy. Cinacalcet was the first allosteric modulator of a G proteincoupled receptor to achieve regulatory approval. This review will focus on the technologies used to discover and develop allosterically acting calcimimetics and calcilytics as novel therapies for bone and mineral-related disorders. PMID:24050279

  14. Toward understanding the molecular basis for chemical allosteric modulator design.

    PubMed

    Wang, Qi; Zheng, Mingyue; Huang, Zhimin; Liu, Xinyi; Zhou, Huchen; Chen, Yingyi; Shi, Ting; Zhang, Jian

    2012-09-01

    Among the regulation mechanisms of cellular function, allosteric regulation is the most direct, rapid and efficient. Due to the wider receptor selectivity and lower target-based toxicity, compared with orthosteric ligands, allosteric modulators are expected to play a larger role in pharmaceutical research and development. However, current difficulties, such as a low affinity and unknown structural features of potential allosteric small-molecules, usually obstruct the discovery of allosteric modulators. In this study, we compared known allosteric modulators with various compounds from different databases to unveil the structural and qualitative characteristics of allosteric modulators. The results show that allosteric modulators generally contain more hydrophobic scaffolds and have a higher structural rigidity, i.e., less rotatable bonds and more rings. Based on this analysis, an empirical rule was defined to determine the structural requirements for an allosteric modulator. It was found that a large proportion of allosteric modulators (80%) can be successfully retrieved by this "allosteric-like" filter, which shows good discriminatory power in identifying allosteric modulators. Therefore, the study provides deeper insight into the chemical properties of allosteric modulators and has a good potential for the design or optimization of allosteric compounds. PMID:23085171

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

    PubMed

    Uteshev, Victor V

    2014-03-15

    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, nicotinic-PAM-based treatments are expected to augment the endogenous cholinergic tone in a spatially and temporally restricted manner creating the potential for differential efficacy and improved safety as compared to exogenous orthosteric nicotinic agonists that activate nicotinic receptors indiscriminately. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs.

  16. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25998276

  18. Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor.

    PubMed

    Rovira, Xavier; Harrak, Youssef; Trapero, Ana; González-Bulnes, Patricia; Malhaire, Fanny; Pin, Jean-Philippe; Goudet, Cyril; Giraldo, Jesús; Llebaria, Amadeu

    2014-12-01

    The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu4 ) positive allosteric modulators (PAMs) with the cyclohexane 1,2-dicarboxylic scaffold present in cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six-membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu4 conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu4 pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu4 allosteric binding site, thus providing a step forward in structure-based drug design for mGlu4 PAMs.

  19. 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.

  20. Neurobiological Insights from mGlu Receptor Allosteric Modulation

    PubMed Central

    O’Brien, Daniel E

    2016-01-01

    Allosteric modulation of metabotropic glutamate (mGlu) receptors offers a promising pharmacological approach to normalize neural circuit dysfunction associated with various psychiatric and neurological disorders. As mGlu receptor allosteric modulators progress through discovery and clinical development, both technical advances and novel tool compounds are providing opportunities to better understand mGlu receptor pharmacology and neurobiology. Recent advances in structural biology are elucidating the structural determinants of mGlu receptor–negative allosteric modulation and supplying the means to resolve active, allosteric modulator-bound mGlu receptors. The discovery and characterization of allosteric modulators with novel pharmacological profiles is uncovering the biological significance of their intrinsic agonist activity, biased mGlu receptor modulation, and novel mGlu receptor heterodimers. The development and exploitation of optogenetic and optopharmacological tools is permitting a refined spatial and temporal understanding of both mGlu receptor functions and their allosteric modulation in intact brain circuits. Together, these lines of research promise to provide a more refined understanding of mGlu receptors and their allosteric modulation that will inform the development of mGlu receptor allosteric modulators as neurotherapeutics in the years to come. PMID:26647381

  1. Allosteric Modulators for mGlu Receptors

    PubMed Central

    Gasparini, F; Spooren, W

    2007-01-01

    The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson’s disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors. PMID:19305801

  2. Oxazolidinone-based allosteric modulators of mGluR5: Defining molecular switches to create a pharmacological tool box.

    PubMed

    Huang, Hong; Degnan, Andrew P; Balakrishnan, Anand; Easton, Amy; Gulianello, Michael; Huang, Yanling; Matchett, Michele; Mattson, Gail; Miller, Regina; Santone, Kenneth S; Senapati, Arun; Shields, Eric E; Sivarao, Digavalli V; Snyder, Lawrence B; Westphal, Ryan; Whiterock, Valerie J; Yang, Fukang; Bronson, Joanne J; Macor, John E

    2016-09-01

    Herein we describe the structure activity relationships uncovered in the pursuit of an mGluR5 positive allosteric modulator (PAM) for the treatment of schizophrenia. It was discovered that certain modifications of an oxazolidinone-based chemotype afforded predictable changes in the pharmacological profile to give analogs with a wide range of functional activities. The discovery of potent silent allosteric modulators (SAMs) allowed interrogation of the mechanism-based liabilities associated with mGluR5 activation and drove our medicinal chemistry effort toward the discovery of low efficacy (fold shift) PAMs devoid of agonist activity. This work resulted in the identification of dipyridyl 22 (BMS-952048), a compound with a favorable free fraction, efficacy in a rodent-based cognition model, and low potential for convulsions in mouse. PMID:27496211

  3. Pharmacological Modulation of NMDA Receptor Activity and the Advent of Negative and Positive Allosteric Modulators

    PubMed Central

    Monaghan, Daniel T.; Irvine, Mark W.; Costa, Blaise Mathias; Fang, Guangyu; Jane, David E.

    2012-01-01

    The NMDA receptor (NMDAR) family of L-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the L-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identifed new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C & GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the L-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics. PMID:22269804

  4. Progress in the developement of positive allosteric modulators of the metabotropic glutamate receptor 2.

    PubMed

    Trabanco, A A; Cid, J M; Lavreysen, H; Macdonald, G J; Tresadern, G

    2011-01-01

    The metabotropic glutamate type 2 (mGlu2) receptor is a G-protein coupled receptor (GPCR) expressed on presynaptic nerve terminals where it negatively modulates glutamate and GABA release. Mixed mGlu2/mGlu3 orthosteric agonists such as LY354740 have shown activity in a range of preclinical animal models of anxiety and schizophrenia. Clinical work with LY354740 demonstrated activity in a CO(2) inhalation study suggesting application in the treatment of anxiety related disorders. Subsequently, a related prodrug LY2140023 demonstrated improvements in positive and negative symptoms in patients suffering from schizophrenia. These molecules exhibit combined mGlu2/mGlu3 activity although there is evidence from knock-out studies that preclinical anti-psychotic effects may be mediated via the mGlu2 receptor. An alternative avenue for modulating GPCRs is to act via allosteric mechanisms, binding at a different site from the orthosteric agonist. Since the first discovery of mGlu2 positive allosteric modulators (PAMs) such as 2,2,2-TEMPS and BINA, multiple families of mGlu2 modulators have been reported and several have entered into clinical development. This review focuses on recent advances in the development of novel mGlu2 PAMs by analysis of compounds disclosed in research articles and patent literature between 2007 and 2010. PMID:21110815

  5. Chemical, Target, and Bioactive Properties of Allosteric Modulation

    PubMed Central

    van Westen, Gerard J. P.; Gaulton, Anna; Overington, John P.

    2014-01-01

    Allosteric modulators are ligands for proteins that exert their effects via a different binding site than the natural (orthosteric) ligand site and hence form a conceptually distinct class of ligands for a target of interest. Here, the physicochemical and structural features of a large set of allosteric and non-allosteric ligands from the ChEMBL database of bioactive molecules are analyzed. In general allosteric modulators are relatively smaller, more lipophilic and more rigid compounds, though large differences exist between different targets and target classes. Furthermore, there are differences in the distribution of targets that bind these allosteric modulators. Allosteric modulators are over-represented in membrane receptors, ligand-gated ion channels and nuclear receptor targets, but are underrepresented in enzymes (primarily proteases and kinases). Moreover, allosteric modulators tend to bind to their targets with a slightly lower potency (5.96 log units versus 6.66 log units, p<0.01). However, this lower absolute affinity is compensated by their lower molecular weight and more lipophilic nature, leading to similar binding efficiency and surface efficiency indices. Subsequently a series of classifier models are trained, initially target class independent models followed by finer-grained target (architecture/functional class) based models using the target hierarchy of the ChEMBL database. Applications of these insights include the selection of likely allosteric modulators from existing compound collections, the design of novel chemical libraries biased towards allosteric regulators and the selection of targets potentially likely to yield allosteric modulators on screening. All data sets used in the paper are available for download. PMID:24699297

  6. 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.

  7. Novel Allosteric Modulators of G Protein-coupled Receptors*

    PubMed Central

    Gentry, Patrick R.; Sexton, Patrick M.; Christopoulos, Arthur

    2015-01-01

    G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization. PMID:26100627

  8. QSAR design of triazolopyridine mGlu2 receptor positive allosteric modulators.

    PubMed

    Tresadern, Gary; Cid, José-Maria; Trabanco, Andrés A

    2014-09-01

    Two QSAR approaches were applied to assist the design and to prioritise the synthesis of new active mGlu2 receptor positive allosteric modulators (PAMs). With the aim to explore a particular point of substitution the models successfully prioritised molecules originating from chemistry ideas and a large virtual library. The two methods, 3D topomer CoMFA and support vector machines with 2D ECFP6 fingerprints, delivered good correlation and success in this prospective application. Fourteen molecules with different substituent decoration were identified by the in silico models and synthesised. They were found to be highly active and their mGlu2 receptor PAM activity (pEC50) was predicted within 0.3 and 0.4log units of error with the two methods. The value of the molecules and the models for the future of the project is discussed. PMID:25086773

  9. Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4

    PubMed Central

    Niswender, Colleen M.; Johnson, Kari A.; Weaver, C. David; Jones, Carrie K.; Xiang, Zixiu; Luo, Qingwei; Rodriguez, Alice L.; Marlo, Joy E.; de Paulis, Tomas; Thompson, Analisa D.; Days, Emily L.; Nalywajko, Tasha; Aust, Cheryl A.; Williams, Michael Baxter; Ayala, Jennifer E.; Williams, Richard; Lindsley, Craig W.; Conn, P. Jeffrey

    2008-01-01

    Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. PHCCC is a positive allosteric modulator (PAM) of mGluR4 which has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency and poor solubility. Via high-throughput screening, we discovered over 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, VU0155041, contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD. PMID:18664603

  10. Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

    PubMed Central

    Gregory, Karen J.

    2015-01-01

    The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

  11. 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

  12. 4-Phenylpyridin-2-one Derivatives: A Novel Class of Positive Allosteric Modulator of the M1 Muscarinic Acetylcholine Receptor.

    PubMed

    Mistry, Shailesh N; Jörg, Manuela; Lim, Herman; Vinh, Natalie B; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Lane, J Robert; Scammells, Peter J

    2016-01-14

    Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer's and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization. PMID:26624844

  13. Discovery and SAR of novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5)

    PubMed Central

    Martín-Martín, María Luz; Bartolomé-Nebreda, José Manuel; Conde-Ceide, Susana; Alonso de Diego, Sergio A.; López, Silvia; Martínez-Viturro, Carlos M.; Tong, Han Min; Lavreysen, Hilde; Macdonald, Gregor J.; Steckler, Thomas; Mackie, Claire; Bridges, Thomas M.; Daniels, J. Scott; Niswender, Colleen M.; Noetzel, Meredith J.; Jones, Carrie K.; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2015-01-01

    We report the discovery and SAR of two novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of several structural features in the western and eastern part of the imidazopyrimidinone core and combinations thereof, revealed compound 4a as a mGlu5 PAM with good in vitro potency and efficacy, acceptable drug metabolism and pharmacokinetic (DMPK) properties and in vivo efficacy in an amphetamine-based model of psychosis. However, the presence of CNS-mediated adverse effects in preclinical species precluded any further in vivo evaluation. PMID:25683622

  14. 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.

  15. 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. PMID:26232639

  16. 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

  17. Allosteric modulators for the treatment of schizophrenia: targeting glutamatergic networks.

    PubMed

    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.

  18. Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release from the endoplasmic reticulum.

    PubMed

    Guerra-Álvarez, María; Moreno-Ortega, Ana J; Navarro, Elisa; Fernández-Morales, José Carlos; Egea, Javier; López, Manuela G; Cano-Abad, María F

    2015-05-01

    Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2

  19. Interaction between positive allosteric modulators and trapping blockers of the NMDA receptor channel

    PubMed Central

    Emnett, Christine M; Eisenman, Lawrence N; Mohan, Jayaram; Taylor, Amanda A; Doherty, James J; Paul, Steven M; Zorumski, Charles F; Mennerick, Steven

    2015-01-01

    Background and Purpose Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers. Experimental Approach We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. Key Results SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks – measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. Conclusions and Implications Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour. PMID:25377730

  20. 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).

  1. Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors.

    PubMed

    Regan, Michael C; Furukawa, Hiro

    2016-09-21

    Two articles in this issue of Neuron (Yelshanskaya et al., 2016; Yi et al., 2016) explore the structural basis of allosteric inhibition in ionotropic glutamate receptors, providing key insights into how iGluRs function in the brain as well as how they might be pharmacologically modulated in neurological disorders and disease. PMID:27657445

  2. 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

    2013-08-01

    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 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.

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

    PubMed Central

    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 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. PMID:23869026

  4. Discovery and SAR of a novel series of metabotropic glutamate receptor 5 positive allosteric modulators with high ligand efficiency

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Bridges, Thomas M.; Vinson, Paige N.; Steckler, Thomas; Lavreysen, Hilde; Mackie, Claire; Bartolomé-Nebreda, José M.; Conde-Ceide, Susana; Tong, Han Min; Macdonald, Gregor J.; Daniels, J. Scott; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    We report the optimization of a series of novel metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from a 5,6-bicyclic class of dihydropyrazolo[1,5-a]pyridin-4(5H)-ones containing a phenoxymethyl linker. Studies focused on a survey of nonamide containing hydrogen bond accepting (HBA) pharmacophore replacements. A highly potent and selective PAM, 2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyridin-4(5H)-one 11, VU0462054), bearing a simple ketone moiety, was identified (LE = 0.52, LELP = 3.2). In addition, hydroxyl, difluoro, ether, and amino variations were examined. Despite promising lead properties and exploration of alternative core heterocycles, linkers, and ketone replacements, oxidative metabolism and in vivo clearance remained problematic for the series. PMID:24961642

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

    PubMed

    Yang, Fukang; Snyder, Lawrence B; Balakrishnan, Anand; Brown, Jeffrey M; Sivarao, Digavalli V; Easton, Amy; Fernandes, Alda; Gulianello, Michael; Hanumegowda, Umesh M; Huang, Hong; Huang, Yanling; Jones, Kelli M; Li, Yu-Wen; Matchett, Michele; Mattson, Gail; Miller, Regina; Santone, Kenneth S; Senapati, Arun; Shields, Eric E; Simutis, Frank J; Westphal, Ryan; Whiterock, Valerie J; Bronson, Joanne J; Macor, John E; Degnan, Andrew P

    2016-03-10

    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

  6. 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

  7. Antidepressant activity in mice elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

    PubMed

    Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Biala, Grazyna; Jozwiak, Krzysztof; Arias, Hugo R

    2014-05-21

    The objective of the current study is to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors (AChRs), produces antidepressant-like behavior in mice, and reactivates desensitized α7 AChRs expressed in CH3-α7 cells. Mice from both sexes were injected (i.p.) with PAM-2 (1.0mg/kg) on a daily basis for three weeks. Forced swim tests (FSTs) were performed on Day 1 and Day 7 to determine the acute and subchronic effects of PAM-2, respectively, and on Days 14 and 21 to determine its chronic activity. To examine the residual effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 28 and 35. Our results indicate that: (1) PAM-2 does not induce acute antidepressant effects in male or female mice, (2) PAM-2 induces antidepressant effects in mice from both sexes after one (subchronic) and two (chronic) weeks, whereas at the third week (chronic), the antidepressant effect is decreased in male and increased in female mice. Since PAM-2 does not influence the locomotor activity of mice, the observed antidepressant activity is not driven by nonspecific motor-stimulant actions, (3) the residual antidepressant effect mediated by PAM-2 after one week of treatment cessation is observed only in female mice, and finally the Ca(2+) influx results indicate that (4) PAM-2 can reactivate desensitized α7 AChRs. Our results clearly indicate that PAM-2 elicits antidepressant activity, probably by enhancing the activity of the endogenous neurotransmitter acetylcholine on α7 AChRs, without inducing receptor desensitization, and that this activity is gender-dependent. This is the first time that an antidepressant activity is described for an α7 PAM, supporting further studies as potential therapeutic medications for depressive states. PMID:24708923

  8. Power penalties for multi-level PAM modulation formats at arbitrary bit error rates

    NASA Astrophysics Data System (ADS)

    Kaliteevskiy, Nikolay A.; Wood, William A.; Downie, John D.; Hurley, Jason; Sterlingov, Petr

    2016-03-01

    There is considerable interest in combining multi-level pulsed amplitude modulation formats (PAM-L) and forward error correction (FEC) in next-generation, short-range optical communications links for increased capacity. In this paper we derive new formulas for the optical power penalties due to modulation format complexity relative to PAM-2 and due to inter-symbol interference (ISI). We show that these penalties depend on the required system bit-error rate (BER) and that the conventional formulas overestimate link penalties. Our corrections to the standard formulas are very small at conventional BER levels (typically 1×10-12) but become significant at the higher BER levels enabled by FEC technology, especially for signal distortions due to ISI. The standard formula for format complexity, P = 10log(L-1), is shown to overestimate the actual penalty for PAM-4 and PAM-8 by approximately 0.1 and 0.25 dB respectively at 1×10-3 BER. Then we extend the well-known PAM-2 ISI penalty estimation formula from the IEEE 802.3 standard 10G link modeling spreadsheet to the large BER case and generalize it for arbitrary PAM-L formats. To demonstrate and verify the BER dependence of the ISI penalty, a set of PAM-2 experiments and Monte-Carlo modeling simulations are reported. The experimental results and simulations confirm that the conventional formulas can significantly overestimate ISI penalties at relatively high BER levels. In the experiments, overestimates up to 2 dB are observed at 1×10-3 BER.

  9. 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.

  10. 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. PMID:17894647

  11. 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

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

    PubMed

    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

  13. Allosteric Modulation of the Calcium-Sensing Receptor

    PubMed Central

    Jensen, Anders A; Bräuner-Osborne, Hans

    2007-01-01

    The calcium (Ca2+)-sensing receptor (CaR) belongs to family C of the G-protein coupled receptors (GPCRs). The receptor is activated by physiological levels of Ca2+ (and Mg2+) and positively modulated by a range of proteinogenic L-α-amino acids. Recently, several synthetic allosteric modulators of the receptor have been developed, which either act as positive modulators (termed calcimimetics) or negative modulators (termed calcilytics). These ligands do not activate the wild-type receptor directly, but rather shift the concentration-response curves of Ca2+ to the left or right, respectively. Like other family C GPCRs, the CaR contains a large amino-terminal domain and a 7-transmembrane domain. Whereas the endogenous ligands for the receptor, Ca2+, Mg2+ and the L-α-amino acids, bind to the amino-terminal domain, most if not all of the synthetic modulators published so far bind to the 7-transmembrane domain. The most prominent physiological function of the CaR is to maintain the extracellular Ca2+ level in a very tight range via control of secretion of parathyroid hormone (PTH). Influence on e.g. secretion of calcitonin from thyroid C-cells and direct action on the tubule of the kidney also contribute to the control of the extracellular Ca2+ level. This control over PTH and Ca2+ levels is partially lost in patients suffering from primary and secondary hyperparathyroidism. The perspectives in CaR as a therapeutic target have been underlined by the recent approval of the calcimimetic cinacalcet for the treatment of certain forms of primary and secondary hyperparathyroidism. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and thus the compound constitutes an important proof-of-concept for future development of allosteric modulators on other GPCR drug targets. PMID:19305800

  14. 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

  15. Tetrahydronaphthyridine and dihydronaphthyridinone ethers as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu₅).

    PubMed

    Turlington, Mark; Malosh, Chrysa; Jacobs, Jon; Manka, Jason T; Noetzel, Meredith J; Vinson, Paige N; Jadhav, Satyawan; Herman, Elizabeth J; Lavreysen, Hilde; Mackie, Claire; Bartolomé-Nebreda, José M; Conde-Ceide, Susana; Martín-Martín, M Luz; Tong, Han Min; López, Silvia; MacDonald, Gregor J; Steckler, Thomas; Daniels, J Scott; Weaver, C David; Niswender, Colleen M; Jones, Carrie K; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

    2014-07-10

    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

  16. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

    PubMed

    Kahsai, Alem W; Wisler, James W; Lee, Jungmin; Ahn, Seungkirl; Cahill Iii, 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

    2016-09-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing 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 through unique, previously unappreciated mechanisms. Here, using 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, the 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

  17. Positive allosteric modulation reveals a specific role for mGlu2 receptors in sensory processing in the thalamus.

    PubMed

    Copeland, C S; Neale, S A; Salt, T E

    2012-02-15

    Group II metabotropic glutamate receptor (mGlu) modulation of sensory processing in the rat ventrobasal thalamic nucleus (VB) has been extensively studied in vivo. However, it is not yet known what the relative contributions are of the Group II mGlu receptor subtypes (mGlu2 and mGlu3) to this modulation, nor to what extent these receptors may be activated under physiological conditions during this process. Using single-neurone recording in the rat VB in vivo with local application of the selective Group II agonist LY354740 and the subtype selective mGlu2 positive allosteric modulator (PAM) LY487379, our findings were twofold. Firstly, we found that there is an mGlu2 component to the effects of LY354740 on sensory responses in the VB. Secondly, we have demonstrated that application of the PAM alone can modulate sensory responses of single neurones in vivo. This indicates that mGlu2 receptors can be activated by endogenous agonist following physiological sensory stimulation. We speculate that the mGlu2 subtype could be activated under physiological stimulus-evoked conditions by 'glutamate spillover' from synapses between excitatory sensory afferents and VB neurones that can lead to a reduction in sensory-evoked inhibition arising from the thalamic reticular nucleus (TRN). We propose that this potential mGlu2 receptor modulation of inhibition could play an important role in discerning relevant information from background activity upon physiological sensory stimulation. Furthermore, this could be a site of action for mGlu2 PAMs to modulate cognitive processes. PMID:22199165

  18. The allosteric modulation of lipases and its possible biological relevance

    PubMed Central

    Köhler, Jens; Wünsch, Bernhard

    2007-01-01

    Background During the development of an enantioselective synthesis using the lipase from Mucor miehei an unusual reaction course was observed, which was analyzed precisely. For the first time an allosteric modulation of a lipase changing its selectivity was shown. Theory Considering the biological relevance of the discovered regulation mechanism we developed a theory that describes the regulation of energy homeostasis and fat metabolism. Conclusion This theory represents a new approach to explain the cause of the metabolic syndrome and provides an innovative basis for further research activity. PMID:17825093

  19. Allosteric modulators of the hERG K(+) channel: radioligand binding assays reveal allosteric characteristics of dofetilide analogs.

    PubMed

    Yu, Zhiyi; Klaasse, Elisabeth; Heitman, Laura H; Ijzerman, Adriaan P

    2014-01-01

    Drugs that block the cardiac K(+) 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(+) 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 [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 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(+) channel, which is discussed in the light of findings on other ion channels. PMID:24200993

  20. 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. PMID:26003081

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

    PubMed Central

    Rodriguez, Alice L.; Zhou, Ya; Williams, Richard; Weaver, C. David; Vinson, Paige N.; Dawson, Eric S.; Steckler, Thomas; Lavreysen, Hilde; Mackieg, 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.

    2012-01-01

    Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu5 PAM chemotype. PMID:23142615

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

    PubMed

    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

    2015-12-01

    The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-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 [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]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.

  3. Effects of alpha-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.

    PubMed

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

    Evidence indicates that microglial activation contributes to the pathophysiology and maintenance of neuroinflammatory pain involving central nervous system alpha-7 nicotinic acetylcholine receptors. The objective of the present study was to determine the effects of 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an alpha-7 nicotinic acetylcholine receptor positive allosteric modulator (PAM), on tactile allodynia and thermal hyperalgesia following lipopolysaccharide (LPS)-induced microglial activation in hippocampus, a neuroinflammatory pain model in mice. In addition, we examined the effects of TQS on microglial activation marker, an ionized calcium-binding adapter molecule 1 (Iba-1), in the hippocampus may be associated with neuroinflammatory pain. Pretreatment of TQS (4mg/kg) significantly reduced LPS (1mg/kg)-induced tactile allodynia and thermal hyperalgesia. Moreover, pretreatment of methyllycaconitine (3mg/kg) significantly reversed TQS-induced antiallodynic and antihyperalgesic responses indicating the involvement of alpha-7 nicotinic acetylcholine receptor. Pretreatment of TQS significantly decreased LPS-induced increased in hippocampal Iba-1 expression. Overall, these results suggest that TQS reduces LPS-induced neuroinflammatory pain like symptoms via modulating microglial activation likely in the hippocampus and/or other brain region by targeting alpha-7 nicotinic acetylcholine receptor. Therefore, alpha-7 nicotinic acetylcholine receptor PAM such as TQS could be a potential drug candidate for the treatment of neuroinflammatory pain.

  4. 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.

  5. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212)

    SciTech Connect

    Tron, V.A.; Coughlin, M.D.; Jang, D.E.; Stanisz, J.; Sauder, D.N. )

    1990-04-01

    Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

  6. Pharmacological and pharmacokinetic properties of JNJ-40411813, a positive allosteric modulator of the mGlu2 receptor

    PubMed Central

    Lavreysen, Hilde; Ahnaou, Abdellah; Drinkenburg, Wilhelmus; Langlois, Xavier; Mackie, Claire; Pype, Stefan; Lütjens, Robert; Le Poul, Emmanuel; Trabanco, Andrés A; Nuñez, José María Cid

    2015-01-01

    Compounds modulating metabotropic glutamate type 2 (mGlu2) receptor activity may have therapeutic benefits in treating psychiatric disorders like schizophrenia and anxiety. The pharmacological and pharmacokinetic properties of a novel mGlu2 receptor-positive allosteric modulator (PAM), 1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-2(1H)-pyridinone (JNJ-40411813/ADX71149) are described here. JNJ-40411813 acts as a PAM at the cloned mGlu2 receptor: EC50 = 147 ± 42 nmol/L in a [35S]GTPγS binding assay with human metabotropic glutamate type 2 (hmGlu2) CHO cells and EC50 = 64 ± 29 nmol/L in a Ca2+ mobilization assay with hmGlu2 Gα16 cotransfected HEK293 cells. [35S]GTPγS autoradiography on rat brain slices confirmed PAM activity of JNJ-40411813 on native mGlu2 receptor. JNJ-40411813 displaced [3H]JNJ-40068782 and [3H]JNJ-46281222 (mGlu2 receptor PAMs), while it failed to displace [3H]LY341495 (a competitive mGlu2/3 receptor antagonist). In rats, JNJ-40411813 showed ex vivo mGlu2 receptor occupancy using [3H]JNJ-46281222 with ED50 of 16 mg/kg (p.o.). PK-PD modeling using the same radioligand resulted in an EC50 of 1032 ng/mL. While JNJ-40411813 demonstrated moderate affinity for human 5HT2A receptor in vitro (Kb = 1.1 μmol/L), higher than expected 5HT2A occupancy was observed in vivo (in rats, ED50 = 17 mg/kg p.o.) due to a metabolite. JNJ-40411813 dose dependently suppressed REM sleep (LAD, 3 mg/kg p.o.), and promoted and consolidated deep sleep. In fed rats, JNJ-40411813 (10 mg/kg p.o.) was rapidly absorbed (Cmax 938 ng/mL at 0.5 h) with an absolute oral bioavailability of 31%. Collectively, our data show that JNJ-40411813 is an interesting candidate to explore the therapeutic potential of mGlu2 PAMs, in in vivo rodents experiments as well as in clinical studies. PMID:25692015

  7. 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

  8. 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. PMID:25893897

  9. 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.

  10. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors – A Structural Perspective of Ligands and Mutants

    PubMed Central

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G.; Weiss, Dahlia; Arsova, Angela; Marshall, Fiona H.; Bräuner-Osborne, Hans; Gloriam, David E.

    2015-01-01

    The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far this has not led to a marketed drug, largely because of the difficulties in achieving subtype-selective compounds with desired properties. Very recently the first crystal structures were published for the transmembrane domain of two metabotropic glutamate receptors in complex with negative allosteric modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different mGlu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs. PMID:26359761

  11. Preclinical evaluation of the antipsychotic potential of the mGlu2-positive allosteric modulator JNJ-40411813

    PubMed Central

    Lavreysen, Hilde; Langlois, Xavier; Donck, Luc Ver; Nuñez, José María Cid; Pype, Stefan; Lütjens, Robert; Megens, Anton

    2015-01-01

    JNJ-40411813/ADX71149 (1-butyl-3-chloro-4-(4-phenylpiperidin-1-yl) pyridin-2(1H)-one) is a positive allosteric modulator (PAM) of the mGlu2 receptor, which also displays 5-Hydroxytryptamine (5HT2A) antagonism after administration in rodents due to a rodent-specific metabolite. JNJ-40411813 was compared with the orthosteric mGlu2/3 agonist LY404039 (4-amino-2-thiabicyclo [3.1.0] hexane-4,6-dicarboxylic acid 2,2-dioxide), the selective mGlu2 PAM JNJ-42153605 (3-(cyclopropylmethyl)-7-(4-phenylpiperidin-1-yl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine) and the 5HT2A antagonist ritanserin in rodent models for antipsychotic activity and potential side effects, attempting to differentiate between the various compounds and mechanisms of action. In mice, JNJ-40411813, JNJ-42153605, and LY404039 inhibited spontaneous locomotion and phencyclidine- and scopolamine-induced but not d-amphetamine-induced hyperlocomotion; the 5HT2A antagonist ritanserin inhibited only spontaneous locomotion and phencyclidine-induced hyperlocomotion. As measured by 2-deoxyglucose uptake, all compounds reversed memantine-induced brain activation in mice. The two mGlu2 PAMs and LY404039, but not ritanserin, inhibited conditioned avoidance behavior in rats. Like ritanserin, the mGlu2 ligands antagonized 2,5-dimethoxy-4-methylamphetamine-induced head twitches in rats. LY404039 but not the mGlu2 PAMs impaired rotarod performance in rats and increased the acoustic startle response in mice. Our results show that although 5HT2A antagonism has effect in some models, mGlu2 receptor activation is sufficient for activity in several animal models of antipsychotic activity. The mGlu2 PAMs mimicked the in vivo pharmacodynamic effects observed with LY404039 except for effects on the rotarod and acoustic startle, suggesting that they produce a primary activity profile similar to that of the mGlu2/3 receptor agonist while they can be differentiated based on their secondary activity profile. The results are

  12. The structural basis of ATP as an allosteric modulator.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-09-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  13. The Structural Basis of ATP as an Allosteric Modulator

    PubMed Central

    Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  14. 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.

  15. 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.

  16. 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.

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

    PubMed

    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

  18. 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

  19. 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.

  20. 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. PMID:27129924

  1. Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.

    PubMed

    Ghosh, Pratiti; Moitra, Karobi; Maki, Nazli; Dey, Saibal

    2006-06-01

    The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.

  2. Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders

    PubMed Central

    Conn, P. Jeffrey; Lindsley, Craig W.; Meiler, Jens; Niswender, Colleen M.

    2014-01-01

    Novel allosteric modulators of G protein-coupled receptors (GPCRs) are providing fundamental advances in the development of GPCR ligands with high subtype selectivity and novel modes of efficacy that have not been possible with traditional approaches. As new allosteric modulators are advancing as drug candidates, we are developing an increased understanding of the major advantages and broad range of activities that can be achieved with these agents through selective modulation of specific signalling pathways, differential effects on GPCR homodimers versus heterodimers, and other properties. This understanding creates exciting opportunities, as well as unique challenges, in the optimization of novel therapeutic agents for disorders of the central nervous system. PMID:25176435

  3. mGluR5 Positive and Negative Allosteric Modulators Differentially Affect Dendritic Spine Density and Morphology in the Prefrontal Cortex.

    PubMed

    LaCrosse, Amber L; Taylor, Sara B; Nemirovsky, Natali E; Gass, Justin T; Olive, Michael F

    2015-01-01

    Positive and negative allosteric modulators (PAMs and NAMs, respectively) of type 5 metabotropic glutamate receptors (mGluR5) are currently being investigated as novel treatments for neuropsychiatric diseases including drug addiction, schizophrenia, and Fragile X syndrome. However, only a handful of studies have examined the effects of mGluR5 PAMs or NAMs on the structural plasticity of dendritic spines in otherwise naïve animals, particularly in brain regions mediating executive function. In the present study, we assessed dendritic spine density and morphology in pyramidal cells of the medial prefrontal cortex (mPFC) after repeated administration of either the prototypical mGluR5 PAM 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5- yl)benzamide (CDPPB, 20 mg/kg), the clinically utilized mGluR5 NAM 1-(3-chlorophenyl)-3-(3-methyl-5-oxo-4Himidazol- 2-yl)urea (fenobam, 20 mg/kg), or vehicle in male Sprague-Dawley rats. Following once daily treatment for 10 consecutive days, coronal brain sections containing the mPFC underwent diolistic labeling and 3D image analysis of dendritic spines. Compared to vehicle treated animals, rats administered fenobam exhibited significant increases in dendritic spine density and the overall frequency of spines with small (<0.2 μm) head diameters, decreases in frequency of spines with medium (0.2-0.4 μm) head diameters, and had no changes in frequency of spines with large head diameters (>0.4 μm). Administration of CDPPB had no discernable effects on dendritic spine density or morphology, and neither CDPPB nor fenobam had any effect on spine length or volume. We conclude that mGluR5 PAMs and NAMs differentially affect mPFC dendritic spine structural plasticity in otherwise naïve animals, and additional studies assessing their effects in combination with cognitive or behavioral tasks are needed.

  4. 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.

  5. Synthesis and biological evaluation of negative allosteric modulators of the Kv11.1(hERG) channel.

    PubMed

    Yu, Zhiyi; van Veldhoven, Jacobus P D; 't Hart, Ingrid M E; Kopf, Adrian H; Heitman, Laura H; IJzerman, Adriaan P

    2015-12-01

    We synthesized and evaluated a series of compounds for their allosteric modulation at the Kv11.1 (hERG) channel. Most compounds were negative allosteric modulators of [(3)H]dofetilide binding to the channel, in particular 7f, 7h-j and 7p. Compounds 7f and 7p were the most potent negative allosteric modulators amongst all ligands, significantly increasing the dissociation rate of dofetilide in the radioligand kinetic binding assay, while remarkably reducing the affinities of dofetilide and astemizole in a competitive displacement assay. Additionally, both 7f and 7p displayed peculiar displacement characteristics with Hill coefficients significantly distinct from unity as shown by e.g., dofetilide, further indicative of their allosteric effects on dofetilide binding. Our findings in this investigation yielded several promising negative allosteric modulators for future functional and clinical research with respect to their antiarrhythmic propensities, either alone or in combination with known Kv11.1 blockers. PMID:26519929

  6. Group I and group II metabotropic glutamate receptor allosteric modulators as novel potential antipsychotics

    PubMed Central

    Walker, Adam G.; Conn, P. Jeffrey

    2014-01-01

    Recently, there has been a shift in the schizophrenia field focusing on restoring glutamate signaling. Extensive preclinical data suggests that mGlu5 PAMs could have efficacy in all three symptom domains but there is concern of potential adverse effects. New insights into mechanisms underlying this toxicity may provide a path for discovery of safe mGlu5 PAMs. Genetic mutations in mGlu1 have been described in schizophrenics creating interest in this receptor as a therapeutic target. Preclinical data demonstrated the antipsychotic potential of of mGlu2/3 agonists but clinical trials were not successful. However, studies have suggested that mGlu2 is the subtype mediating antipsychotic effects and selective mGlu2 PAMs are now in clinical development. Finally, recent genetic studies suggest mGlu3 modulators may be pro-cognitive. PMID:25462291

  7. Group I and group II metabotropic glutamate receptor allosteric modulators as novel potential antipsychotics.

    PubMed

    Walker, Adam G; Conn, P Jeffrey

    2015-02-01

    Recently, there has been a shift in the schizophrenia field focusing on restoring glutamate signaling. Extensive preclinical data suggests that mGlu5 PAMs could have efficacy in all three symptom domains but there is concern of potential adverse effects. New insights into mechanisms underlying this toxicity may provide a path for discovery of safe mGlu5 PAMs. Genetic mutations in mGlu1 have been described in schizophrenics creating interest in this receptor as a therapeutic target. Preclinical data demonstrated the antipsychotic potential of mGlu2/3 agonists but clinical trials were not successful. However, studies have suggested that mGlu2 is the subtype mediating antipsychotic effects and selective mGlu2 PAMs are now in clinical development. Finally, recent genetic studies suggest mGlu3 modulators may be pro-cognitive. PMID:25462291

  8. 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

  9. 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

  10. Methods for evaluation of positive allosteric modulators of glutamate AMPA receptors.

    PubMed

    Siuda, Edward R; Quirk, Jennifer C; Nisenbaum, Eric S

    2007-01-01

    Hypofunctioning of glutamate synaptic transmission in the central nervous system (CNS) has been proposed as a factor that may contribute to cognitive deficits associated with various neurological and psychiatric disorders. Positive allosteric modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) subtype of glutamate receptors has been proposed as a novel therapeutic approach, because these receptors mediate the majority of rapid excitatory neurotransmission and are intimately involved in long-term changes in synaptic plasticity thought to underlie mnemonic processing. By definition, positive allosteric modulators do not affect AMPA receptor activity alone but can markedly enhance ion flux through the ion channel pore in the presence of bound agonist. Despite this commonality, positive allosteric modulators can be segregated on the basis of the preferential effects on AMPA receptor subunits, their alternatively spliced variants and/or their biophysical mechanism of action. This chapter provides a detailed description of the methodologies used to evaluate the potency/efficacy and biophysical mechanism of action of positive allosteric modulators of AMPA receptors.

  11. Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators.

    PubMed

    Gregory, Karen J; Velagaleti, Ranganadh; Thal, David M; Brady, Ryan M; Christopoulos, Arthur; Conn, P Jeffrey; Lapinsky, David J

    2016-07-15

    G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively "tuning" GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand-GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu5 NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu5 via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu5-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu5 and negative cooperativity with glutamate, interacted with the "common allosteric-binding site," displayed slow binding kinetics, and could irreversibly label mGlu5 following UV exposure. We depleted the number of functional mGlu5 receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu5 allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily. PMID:27115427

  12. Orally Active Metabotropic Glutamate Subtype 2 Receptor Positive Allosteric Modulators: Structure-Activity Relationships and Assessment in a Rat Model of Nicotine Dependence

    PubMed Central

    Sidique, Shyama; Dhanya, Raveendra-Panickar; Sheffler, Douglas J.; Nickols, Hilary Highfield; Yang, Li; Dahl, Russell; Mangravita-Novo, Arianna; Smith, Layton H.; D’Souza, Manoranjan S.; Semenova, Svetlana; Conn, P. Jeffrey; Markou, Athina; Cosford, Nicholas D. P.

    2012-01-01

    Compounds that modulate metabotropic glutamate subtype 2 (mGlu2) receptors have the potential to treat several disorders of the central nervous system (CNS) including drug dependence. Herein we describe the synthesis and structure-activity relationship (SAR) studies around a series of mGlu2 receptor positive allosteric modulators (PAMs). The effects of N-substitution (R1) and substitutions on the aryl ring (R2) were identified as key areas for SAR exploration (Figure 3). Investigation of the effects of varying substituents in both the isoindolinone (2) and benzisothiazolone (3) series led to compounds with improved in vitro potency and/or efficacy. In addition, several analogues exhibited promising pharmacokinetic (PK) properties. Furthermore, compound 2 was shown to dose-dependently decrease nicotine self-administration in rats following oral administration. Our data, showing for the first time efficacy of an mGlu2 receptor PAM in this in vivo model, suggest potential utility for the treatment of nicotine dependence in humans. PMID:23009245

  13. 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

  14. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

    PubMed Central

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-01-01

    Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. PMID:25040887

  15. Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.

    PubMed

    Mony, Laetitia; Zhu, Shujia; Carvalho, Stéphanie; Paoletti, Pierre

    2011-06-17

    NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.

  16. G Protein-Coupled Receptor Heteromerization: A Role in Allosteric Modulation of Ligand BindingS⃞

    PubMed Central

    Gomes, Ivone; IJzerman, Adriaan P.; Ye, Kai; Maillet, Emeline L.

    2011-01-01

    It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study, we examined this possibility by using an established model system of a receptor heteromer consisting of μ and δ opioid receptors. We examined the effect of a number of μ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled δ receptor agonist, [3H]deltorphin II. We also examined the effect of δ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled μ receptor agonist, [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin ([3H]DAMGO). We show that μ receptor ligands are capable of allosterically enhancing δ receptor radioligand binding and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity. PMID:21415307

  17. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator#

    PubMed Central

    Wu, Huixian; Wang, Chong; Gregory, Karen J.; Han, Gye Won; Cho, Hyekyung P.; Xia, Yan; Niswender, Colleen M.; Katritch, Vsevolod; Meiler, Jens; Cherezov, Vadim; Conn, P. Jeffrey; Stevens, Raymond C.

    2014-01-01

    The excitatory neurotransmitter glutamate induces modulatory actions via the metabotropic glutamate receptors (mGlus), which are class C G protein-coupled receptors (GPCRs). We determined the 2.8 Å resolution structure of the human mGlu1 receptor seven-transmembrane (7TM) domain bound to a negative allosteric modulator FITM. The modulator binding site partially overlaps with the orthosteric binding sites of class A GPCRs, but is more restricted compared to most other GPCRs. We observed a parallel 7TM dimer, mediated by cholesterols, suggesting that signaling initiated by glutamate’s interaction with the extracellular domain might be mediated via 7TM interactions within the full-length receptor dimer. A combination of crystallography, structure-activity relationships, mutagenesis, and full-length dimer modeling provides insights on the allosteric modulation and activation mechanism of class C GPCRs. PMID:24603153

  18. Dynamic Modulation of DNA Hybridization Using Allosteric DNA Tetrahedral Nanostructures.

    PubMed

    Song, Ping; Li, Min; Shen, Juwen; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Wang, Lihua; Shi, Jiye; Song, Shiping; Wang, Lianhui; Fan, Chunhai; Zuo, Xiaolei

    2016-08-16

    The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes. PMID:27435955

  19. 13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase*

    PubMed Central

    Kudalkar, Shalley N.; Nikas, Spyros P.; Kingsley, Philip J.; Xu, Shu; Galligan, James J.; Rouzer, Carol A.; Banerjee, Surajit; Ji, Lipin; Eno, Marsha R.; Makriyannis, Alexandros; Marnett, Lawrence J.

    2015-01-01

    Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism. PMID:25648895

  20. 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

  1. Effects of the metabotropic glutamate receptor 5 positive allosteric modulator CDPPB on rats tested with the paired associates learning task in touchscreen-equipped operant conditioning chambers.

    PubMed

    Lins, Brittney R; Howland, John G

    2016-03-15

    Effective treatments for the cognitive symptoms of schizophrenia are critically needed. Positive allosteric modulation (PAM) of metabotropic glutamate receptor subtype 5 (mGluR5) is one strategy currently under investigation to improve these symptoms. Examining cognition using touchscreen-equipped operant chambers may increase translation between preclinical and clinical research through analogous behavioral testing paradigms in rodents and humans. We used acute CDPPB (1-30mg/kg) treatment to examine the effects of mGluR5 PAM in the touchscreen paired associates learning (PAL) task using well-trained rats with and without co-administration of acute MK-801 (0.15mg/kg). CDPPB had no consistent effects on task performance when administered alone and failed to reverse the MK-801 induced impairments at any of the examined doses. Overall, the disruptive effects of MK-801 on PAL were consistent with previous research but increasing mGluR5 signaling is not beneficial in the PAL task. Future research should test whether administration of CDPPB during PAL acquisition increases performance.

  2. The mGluR5 positive allosteric modulator, CDPPB, ameliorates pathology and phenotypic signs of a mouse model of Huntington's disease.

    PubMed

    Doria, J G; de Souza, J M; Andrade, J N; Rodrigues, H A; Guimaraes, I M; Carvalho, T G; Guatimosim, C; Dobransky, T; Ribeiro, F M

    2015-01-01

    Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin protein (htt), leading to motor dysfunction, cognitive decline, psychiatric alterations, and death. The metabotropic glutamate receptor 5 (mGluR5) has been implicated in HD and we have recently demonstrated that mGluR5 positive allosteric modulators (PAMs) are neuroprotective in vitro. In the present study we demonstrate that the mGluR5 PAM, CDPPB, is a potent neuroprotective drug, in vitro and in vivo, capable of delaying HD-related symptoms. The HD mouse model, BACHD, exhibits many HD features, including neuronal cell loss, htt aggregates, motor incoordination and memory impairment. However, chronic treatment of BACHD mice with CDPPB 1.5 mg/kg s.c. for 18 weeks increased the activation of cell signaling pathways important for neuronal survival, including increased AKT and ERK1/2 phosphorylation and augmented the BDNF mRNA expression. CDPPB chronic treatment was also able to prevent the neuronal cell loss that takes place in the striatum of BACHD mice and decrease htt aggregate formation. Moreover, CDPPB chronic treatment was efficient to partially ameliorate motor incoordination and to rescue the memory deficit exhibited by BACHD mice. Importantly, no toxic effects or stereotypical behavior were observed upon CDPPB chronic treatment. Thus, CDPPB is a potential drug to treat HD, preventing neuronal cell loss and htt aggregate formation and delaying HD symptoms.

  3. A type-II positive allosteric modulator of α7 nAChRs reduces brain injury and improves neurological function after focal cerebral ischemia in rats.

    PubMed

    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. Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors.

    PubMed

    Rovira, Xavier; Malhaire, Fanny; Scholler, Pauline; Rodrigo, Jordi; Gonzalez-Bulnes, Patricia; Llebaria, Amadeu; Pin, Jean-Philippe; Giraldo, Jesús; Goudet, Cyril

    2015-01-01

    Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes. PMID:25342125

  5. 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

  6. The mGluR5 Positive Allosteric Modulator CDPPB Does Not Alter Extinction or Contextual Reinstatement of Methamphetamine-Seeking Behavior in Rats.

    PubMed

    Widholm, John J; Gass, Justin T; Cleva, Richard M; Olive, M Foster

    2011-12-24

    Extinction of drug-seeking behavior is a form of new and active learning. Facilitation of extinction learning is of clinical interest since cue exposure therapies for the treatment of addiction have largely been unsuccessful in preventing relapse, primarily due to the context specificity of extinction learning. Recently, several studies have shown that potentiation of glutamatergic transmission can facilitate extinction learning in rodent models of cocaine addiction. In this study we investigated the effects of the type 5 metabotropic glutamate receptor (mGluR5) positive allosteric modulator (PAM) 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the extinction and contextual reinstatement of methamphetamine-seeking behavior. Rats were trained and allowed to self-administer methamphetamine (0.1 mg/kg/infusion) in 2 hr daily sessions in Context A where self-administration chambers had distinct tactile, visual, auditory, and olfactory cues. Next, CDPPB (60 mg/kg) or vehicle was administered prior to subsequent extinction training sessions that were conducted in modified self-administration chambers (Context B) that were Context A. Following 16 days of extinction training in Context B, animals were placed back in Context A for assessment of contextual reinstatement of methamphetamine-seeking behavior. CDPPB failed to produce significant reductions in extinction responding or in the magnitude of contextual reinstatement of methamphetamine-seeking compared to vehicle treated controls. We postulate that numerous factors, including methamphetamine-induced changes in mGluR5 receptor expression or function, may have contributed to the observed lack of effects. Although these findings initially suggest that mGluR5 PAMs may be ineffective in facilitating extinction learning or preventing context-induced relapse in methamphetamine addiction, additional studies are warranted examining effects of other mGluR5 PAMs, particularly those with improved pharmacological

  7. The mGluR5 Positive Allosteric Modulator CDPPB Does Not Alter Extinction or Contextual Reinstatement of Methamphetamine-Seeking Behavior in Rats

    PubMed Central

    Widholm, John J.; Gass, Justin T.; Cleva, Richard M.; Olive, M. Foster

    2012-01-01

    Extinction of drug-seeking behavior is a form of new and active learning. Facilitation of extinction learning is of clinical interest since cue exposure therapies for the treatment of addiction have largely been unsuccessful in preventing relapse, primarily due to the context specificity of extinction learning. Recently, several studies have shown that potentiation of glutamatergic transmission can facilitate extinction learning in rodent models of cocaine addiction. In this study we investigated the effects of the type 5 metabotropic glutamate receptor (mGluR5) positive allosteric modulator (PAM) 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the extinction and contextual reinstatement of methamphetamine-seeking behavior. Rats were trained and allowed to self-administer methamphetamine (0.1 mg/kg/infusion) in 2 hr daily sessions in Context A where self-administration chambers had distinct tactile, visual, auditory, and olfactory cues. Next, CDPPB (60 mg/kg) or vehicle was administered prior to subsequent extinction training sessions that were conducted in modified self-administration chambers (Context B) that were Context A. Following 16 days of extinction training in Context B, animals were placed back in Context A for assessment of contextual reinstatement of methamphetamine-seeking behavior. CDPPB failed to produce significant reductions in extinction responding or in the magnitude of contextual reinstatement of methamphetamine-seeking compared to vehicle treated controls. We postulate that numerous factors, including methamphetamine-induced changes in mGluR5 receptor expression or function, may have contributed to the observed lack of effects. Although these findings initially suggest that mGluR5 PAMs may be ineffective in facilitating extinction learning or preventing context-induced relapse in methamphetamine addiction, additional studies are warranted examining effects of other mGluR5 PAMs, particularly those with improved pharmacological

  8. Muscarinic and Nicotinic Acetylcholine Receptor Agonists and Allosteric Modulators for the Treatment of Schizophrenia

    PubMed Central

    Jones, Carrie K; Byun, Nellie; Bubser, Michael

    2012-01-01

    Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia. PMID:21956443

  9. 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

  10. 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.

  11. First Demonstration of Positive Allosteric-like Modulation at the Human Wild Type Translocator Protein (TSPO).

    PubMed

    Narlawar, Rajeshwar; Werry, Eryn L; Scarf, Alana M; Hanani, Raphy; Chua, Sook Wern; King, Victoria A; Barron, Melissa L; Martins, Ralph N; Ittner, Lars M; Rendina, Louis M; Kassiou, Michael

    2015-11-12

    We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.

  12. Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABAA receptors supports an allosteric model of modulation

    PubMed Central

    Downing, Scott S; Lee, Yan T; Farb, David H; Gibbs, Terrell T

    2005-01-01

    Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABAA receptors, the mechanism of modulation is not well understood. The applicability of an allosteric model with two binding sites for γ-aminobutyric acid (GABA) and one for a BZD-like modulator was investigated. This model predicts that BZDs should enhance the efficacy of partial agonists. Consistent with this prediction, diazepam increased the efficacy of the GABAA receptor partial agonist kojic amine in chick spinal cord neurons. To further test the validity of the model, the effects of diazepam, flurazepam, and zolpidem were examined using wild-type and spontaneously active mutant α1(L263S)β3γ2 GABAA receptors expressed in HEK-293 cells. In agreement with the predictions of the allosteric model, all three modulators acted as direct agonists for the spontaneously active receptors. The results indicate that BZD-like modulators enhance the amplitude of the GABA response by stabilizing the open channel active state relative to the inactive state by less than 1 kcal, which is similar to the energy of stabilization conferred by a single hydrogen bond. PMID:15912137

  13. A question of balance--positive versus negative allosteric modulation of GABA(A) receptor subtypes as a driver of analgesic efficacy in rat models of inflammatory and neuropathic pain.

    PubMed

    Munro, Gordon; Erichsen, Helle K; Rae, Mark G; Mirza, Naheed R

    2011-01-01

    After injury GABA(A) receptor positive allosteric modulators (PAMs) mediate robust analgesia in animals via putative restoration of post-synaptic GABA(A)-α2 and -α3 receptor function within the spinal cord. GABA can also act at GABA(A) receptors localized on primary afferent neurones to inhibit presynaptic neurotransmitter release and produce analgesia via a process called primary afferent depolarization (PAD). Some forms of injury might sufficiently enhance PAD to shift it into a net excitatory process. Thus, negative allosteric modulators (NAMs) might also possess analgesic activity. We have compared compounds capable of either positively or negatively modulating GABA(A) receptors in rat models associated with injury-induced central sensitization. The subtype-selective PAMs NS11394 (1-10 mg/kg) and TPA023 (3-30 mg/kg) attenuated formalin-induced nocifensive behaviours. Similarly, both compounds reversed hindpaw mechanical hypersensitivity and weight bearing deficits in carrageenan-inflamed and nerve-injured rats. The non-selective PAM diazepam (1-5 mg/kg) was ineffective in all models. Surprisingly, both the non-selective NAM FG-7142 (3-30 mg/kg) and the α5-selective NAM α5IA-II (10-60 mg/kg) also attenuated formalin-induced nocifensive behaviours. In carrageenan-inflamed rats α5IA-II reversed mechanical hypersensitivity and weight bearing deficits whilst FG-7142 only attenuated weight bearing deficits. This picture was essentially reversed in nerve-injured rats for these two NAMs. With the exception of NS11394, all compounds attenuated exploratory motility behaviour in rats, either as a consequence of sedative or anxiogenic-like side-effects. These data indicate that the preferred selectivity and activity profiles for mediating analgesia upon activation of GABA(A) receptors might be more complex than previously anticipated, and is worthy of further exploration.

  14. Structural basis for DNA-mediated allosteric regulation facilitated by the AAA+ module of Lon protease.

    PubMed

    Lee, Alan Yueh-Luen; Chen, Yu-Da; Chang, Yu-Yung; Lin, Yu-Ching; Chang, Chi-Fon; Huang, Shing-Jong; Wu, Shih-Hsiung; Hsu, Chun-Hua

    2014-02-01

    Lon belongs to a unique group of AAA+ proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease from Brevibacillus thermoruber (Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+ module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain-domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+ module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

  15. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    PubMed

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  16. Structural Basis for Negative Allosteric Modulation of GluN2A-Containing NMDA Receptors.

    PubMed

    Yi, Feng; Mou, Tung-Chung; Dorsett, Katherine N; Volkmann, Robert A; Menniti, Frank S; Sprang, Stephen R; Hansen, Kasper B

    2016-09-21

    NMDA receptors mediate excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. Here, we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo state of the GluN1 ligand-binding domain (LBD), which is incapable of triggering channel gating. We describe structural determinants of NAM binding in crystal structures of the GluN1/2A LBD heterodimer, and analyses of NAM-bound LBD structures corresponding to active and inhibited receptor states reveal a molecular switch in the modulatory binding site that mediate the allosteric inhibition. NAM binding causes displacement of a valine in GluN2A and the resulting steric effects can be mitigated by the transition from glycine bound to apo state of the GluN1 LBD. This work provides mechanistic insight to allosteric NMDA receptor inhibition, thereby facilitating the development of novel classes NMDA receptor modulators as therapeutic agents. PMID:27618671

  17. Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias.

    PubMed

    Sengmany, K; Gregory, K J

    2016-10-01

    The metabotropic glutamate receptor subtype 5 (mGlu5 ) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  18. Discovery and Development of Small Molecule Allosteric Modulators of Glycoprotein Hormone Receptors

    PubMed Central

    Nataraja, Selvaraj G.; Yu, Henry N.; Palmer, Stephen S.

    2015-01-01

    Glycoprotein hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are heterodimeric proteins with a common α-subunit and hormone-specific β-subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G protein-coupled receptors. FSH receptor (FSHR) and LH receptor are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women and men, respectively. TSH receptor is expressed in thyroid cells and regulates the secretion of T3 and T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients, thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSHR and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical application as demonstrated in preclinical models

  19. Behavioral effects of the cannabinoid CB1 receptor allosteric modulator ORG27569 in rats

    PubMed Central

    Ding, Yuanyuan; Qiu, Yanyan; Jing, Li; Thorn, David A; Zhang, Yanan; Li, Jun-Xu

    2014-01-01

    The cannabinoid CB1 receptor system is involved in feeding behaviors and the CB1 receptor antagonist SR141716A is an effective antiobesity drug. However, SR141716A also has serious side effects, which prompted the exploration of alternative strategies to modulate this important drug target. Recently a CB1 receptor allosteric modulating site has been discovered and the allosteric modulating activity of several modulators including ORG27569 has been characterized in vitro. Yet, little is known of the in vivo pharmacological effects of ORG27569. This study examined the behavioral pharmacology of ORG27569 in rats. ORG27569 (3.2–10 mg/kg, i.p.) selectively attenuated the hypothermic effects of CB1 receptor agonists CP55940 (0.1–1 mg/kg) and anandamide (3.2–32 mg/kg). In contrast, SR141716A only attenuated the hypothermic effects of CP55940 but not anandamide. SR141716A but not ORG27569 blocked CP55940-induced catalepsy and antinociception. In addition, ORG27569 did not modify SR141716A-elicited grooming and scratching behaviors. In feeding studies, ORG27569 decreased palatable and plain food intake which was partially blocked by CP55940. The hypophagic effect of ORG27569 developed tolerance after 4 days of daily 5.6 mg/kg treatment; however, the effect on body weight gain outlasted the drug treatment for 10 days. These data suggest that ORG27569 may not function as a CB1 receptor allosteric modulator in vivo, although its hypophagic activity still has potential therapeutic utility. PMID:25431655

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

    PubMed

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

    2015-11-27

    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.

  1. mGluR5 Positive and Negative Allosteric Modulators Differentially Affect Dendritic Spine Density and Morphology in the Prefrontal Cortex.

    PubMed

    LaCrosse, Amber L; Taylor, Sara B; Nemirovsky, Natali E; Gass, Justin T; Olive, Michael F

    2015-01-01

    Positive and negative allosteric modulators (PAMs and NAMs, respectively) of type 5 metabotropic glutamate receptors (mGluR5) are currently being investigated as novel treatments for neuropsychiatric diseases including drug addiction, schizophrenia, and Fragile X syndrome. However, only a handful of studies have examined the effects of mGluR5 PAMs or NAMs on the structural plasticity of dendritic spines in otherwise naïve animals, particularly in brain regions mediating executive function. In the present study, we assessed dendritic spine density and morphology in pyramidal cells of the medial prefrontal cortex (mPFC) after repeated administration of either the prototypical mGluR5 PAM 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5- yl)benzamide (CDPPB, 20 mg/kg), the clinically utilized mGluR5 NAM 1-(3-chlorophenyl)-3-(3-methyl-5-oxo-4Himidazol- 2-yl)urea (fenobam, 20 mg/kg), or vehicle in male Sprague-Dawley rats. Following once daily treatment for 10 consecutive days, coronal brain sections containing the mPFC underwent diolistic labeling and 3D image analysis of dendritic spines. Compared to vehicle treated animals, rats administered fenobam exhibited significant increases in dendritic spine density and the overall frequency of spines with small (<0.2 μm) head diameters, decreases in frequency of spines with medium (0.2-0.4 μm) head diameters, and had no changes in frequency of spines with large head diameters (>0.4 μm). Administration of CDPPB had no discernable effects on dendritic spine density or morphology, and neither CDPPB nor fenobam had any effect on spine length or volume. We conclude that mGluR5 PAMs and NAMs differentially affect mPFC dendritic spine structural plasticity in otherwise naïve animals, and additional studies assessing their effects in combination with cognitive or behavioral tasks are needed. PMID:25921744

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

    PubMed

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

    2015-11-27

    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

  3. mGluR5 Positive and Negative Allosteric Modulators Differentially Affect Dendritic Spine Density and Morphology in the Prefrontal Cortex

    PubMed Central

    LaCrosse, Amber L.; Taylor, Sara B.; Nemirovsky, Natali E.; Gass, Justin T.; Olive, M. Foster

    2015-01-01

    Positive and negative allosteric modulators (PAMs and NAMs, respectively) of type 5 metabotropic glutamate receptors (mGluR5) are currently being investigated as novel treatments for neuropsychiatric diseases including drug addiction, schizophrenia, and Fragile X syndrome. However, only a handful of studies have examined the effects of mGluR5 PAMs or NAMs on the structural plasticity of dendritic spines in otherwise naïve animals, particularly in brain regions mediating executive function. In the present study, we assessed dendritic spine density and morphology in pyramidal cells of the medial prefrontal cortex (mPFC) after repeated administration of either the prototypical mGluR5 PAM 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, 20 mg/kg), the clinically utilized mGluR5 NAM 1-(3-chlorophenyl)-3-(3-methyl-5-oxo-4H-imidazol-2-yl)urea (fenobam, 20 mg/kg), or vehicle in male Sprague-Dawley rats. Following once daily treatment for 10 consecutive days, coronal brain sections containing the mPFC underwent diolistic labeling and 3D image analysis of dendritic spines. Compared to vehicle treated animals, rats administered fenobam exhibited significant increases in dendritic spine density and the overall frequency of spines with small (<0.2 μm) head diameters, decreases in frequency of spines with medium (0.2–0.4 μm) head diameters, and had no changes in frequency of spines with large head diameters (>0.4 μm). Administration of CDPPB had no discernable effects on dendritic spine density or morphology, and neither CDPPB nor fenobam had any effect on spine length or volume. We conclude that mGluR5 PAMs and NAMs differentially affect mPFC dendritic spine structural plasticity in otherwise naïve animals, and additional studies assessing their effects in combination with cognitive or behavioral tasks are needed. PMID:25921744

  4. The novel metabotropic glutamate receptor 2 positive allosteric modulator, AZD8529, decreases nicotine self-administration and relapse in squirrel monkeys

    PubMed Central

    Justinova, Zuzana; Panlilio, Leigh V.; Secci, Maria E.; Redhi, Godfrey H.; Schindler, Charles W.; Cross, Alan J.; Mrzljak, Ladislav; Medd, Amy; Shaham, Yavin; Goldberg, Steven R.

    2015-01-01

    Background Based on rodent studies, Group II metabotropic glutamate receptors (mGluR2 & 3) were suggested as targets for addiction treatment. However, LY379268 and other Group II agonists do not discriminate between the mainly presynaptic inhibitory mGluR2 (the proposed treatment target) and mGluR3. These agonists also produce tolerance over repeated administration and are no longer considered for addiction treatment. Here, we determined the effects of AZD8529, a selective positive allosteric modulator (PAM) of mGluR2, on abuse-related effects of nicotine in squirrel monkeys and rats. Methods We first assessed modulation of mGluR2 function by AZD8529 using functional in-vitro assays in both membranes prepared from a cell line expressing human mGluR2 and in primate brain slices. We then determined AZD8529 (0.03-10 mg/kg, i.m.) effects on intravenous nicotine self-administration and reinstatement of nicotine seeking induced by nicotine priming or nicotine-associated cues. We also determined AZD8529 effects on food self-administration in monkeys and nicotine-induced dopamine release in accumbens shell in rats. Results AZD8529 potentiated agonist-induced activation of mGluR2 in both the membrane-binding assay and in primate cortex, hippocampus, and striatum. In monkeys, AZD8529 decreased nicotine self-administration at doses (0.3-3 mg/kg) that did not affect food self-administration. AZD8529 also reduced nicotine priming- and cue-induced reinstatement of nicotine seeking after extinction of the drug-reinforced responding. In rats, AZD8529 decreased nicotine-induced accumbens dopamine release. Conclusions Our results provide evidence for efficacy of PAMs of mGluR2 in non-human primate models of nicotine reinforcement and relapse. We propose that this drug class should be considered for nicotine addiction treatment. PMID:25802079

  5. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  6. Recent progress in the synthesis and characterization of group II metabotropic glutamate receptor allosteric modulators.

    PubMed

    Sheffler, Douglas J; Pinkerton, Anthony B; Dahl, Russell; Markou, Athina; Cosford, Nicholas D P

    2011-08-17

    Group II metabotropic glutamate (mGlu) receptors consist of the metabotropic glutamate 2 (mGlu(2)) and metabotropic glutamate 3 (mGlu(3)) receptor subtypes which modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Excessive accumulation of glutamate in the perisynaptic extracellular region triggers mGlu(2) and mGlu(3) receptors to inhibit further release of glutamate. There is growing evidence that the modulation of glutamatergic neurotransmission by small molecule modulators of Group II mGlu receptors has significant potential for the treatment of several neuropsychiatric and neurodegenerative diseases. This review provides an overview of recent progress on the synthesis and pharmacological characterization of positive and negative allosteric modulators of the Group II mGlu receptors. PMID:22860167

  7. Recent Progress in the Synthesis and Characterization of Group II Metabotropic Glutamate Receptor Allosteric Modulators

    PubMed Central

    2011-01-01

    Group II metabotropic glutamate (mGlu) receptors consist of the metabotropic glutamate 2 (mGlu2) and metabotropic glutamate 3 (mGlu3) receptor subtypes which modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Excessive accumulation of glutamate in the perisynaptic extracellular region triggers mGlu2 and mGlu3 receptors to inhibit further release of glutamate. There is growing evidence that the modulation of glutamatergic neurotransmission by small molecule modulators of Group II mGlu receptors has significant potential for the treatment of several neuropsychiatric and neurodegenerative diseases. This review provides an overview of recent progress on the synthesis and pharmacological characterization of positive and negative allosteric modulators of the Group II mGlu receptors. PMID:22860167

  8. Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

    PubMed

    Mony, Laetitia; Kew, James N C; Gunthorpe, Martin J; Paoletti, Pierre

    2009-08-01

    N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.

  9. 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).

  10. Gene modules associated with breast cancer distant metastasis-free survival in the PAM50 molecular subtypes

    PubMed Central

    Liu, Rong; Zhang, Wei; Liu, Zhao-Qian; Zhou, Hong-Hao

    2016-01-01

    To identify PAM50 subtype–specific associations between distant metastasis-free survival (DMFS) in breast cancer (BC) patients and gene modules describing potentially targetable oncogenic pathways, a comprehensive analysis evaluating the prognostic efficacy of published gene signatures in 2027 BC patients from 13 studies was conducted. We calculated 21 gene modules and computed hazard ratios (HRs) for DMFS for one-unit increases in module score, with and without adjustment for clinical characteristics. By comparing gene expression to survival outcomes, we derived four subtype-specific prognostic signatures for BC. Univariate and multivariate analyses showed that in the luminal A subgroup, E2F3, PTEN and GGI gene module scores were associated with clinical outcome. In the luminal B tumors, RAS was associated with DMFS and in the basal-like tumors, ER was associated with DMFS. Our defined gene modules predicted high-risk patients in multivariate analyses for the basal-like (HR: 2.19, p=2.5×10−4), luminal A (HR: 3.03, p=7.2×10−5), luminal B (HR: 3.00, p=2.4×10−10) and HER2+ (HR: 5.49, p=9.7×10−10) subgroups. We found that different modules are associated with DMFS in different BC subtypes. The results of this study could help to identify new therapeutic strategies for specific molecular subgroups of BC, and could enhance efforts to improve patient-specific therapy options. PMID:26934123

  11. 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

  12. Discovery of potent positive allosteric modulators of the α3β2 nicotinic acetylcholine receptor by a chemical space walk in ChEMBL.

    PubMed

    Bürgi, Justus J; Awale, Mahendra; Boss, Silvan D; Schaer, Tifany; Marger, Fabrice; Viveros-Paredes, Juan M; Bertrand, Sonia; Gertsch, Jürg; Bertrand, Daniel; Reymond, Jean-Louis

    2014-05-21

    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

  13. 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

  14. 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.

  15. ADX71441, a novel, potent and selective positive allosteric modulator of the GABAB receptor, shows efficacy in rodent models of overactive bladder

    PubMed Central

    Kalinichev, M; Palea, S; Haddouk, H; Royer-Urios, I; Guilloteau, V; Lluel, P; Schneider, M; Saporito, M; Poli, S

    2014-01-01

    Background and Purpose The GABAB receptor agonist baclofen reduces urethral resistance and detrusor overactivity in patients with spasticity. However, baclofen's side effects limit its use for the treatment of overactive bladder (OAB). Here, we tested a novel GABAB positive allosteric modulator (PAM) ADX71441 in models of OAB in mice and guinea pigs. Experimental Approach Mice were left untreated or given (p.o.) vehicle (1= CMC), ADX71441 (1, 3, 10 mg kg−1) or oxybutynin (100 mg kg−1; Experiment 1) or vehicle (1= CMC), baclofen (1, 3, 6 mg kg−1) or oxybutynin (Experiment 2). Treated mice were then overhydrated with water, challenged with furosemide, before being placed into micturition chambers and monitored for urinary parameters. In anaesthetized guinea pigs, intravesical infusion of acetic acid was used to induce OAB and the effects of ADX71441 (1, 3 mg kg−1) or baclofen (1 mg kg−1), administered i.v., on cystometric parameters were monitored. Key Results In mice, 10 mg kg−1 ADX71441 increased urinary latencies, reduced the number of urinary events and the total and average urinary volumes. In guinea pigs, ADX71441 (1 and 3 mg kg−1) increased the intercontraction interval (ICI) and bladder capacity (BC), and reduced micturition frequency (MF) compared to vehicle. At 3 mg kg−1 ADX71441 completely inhibited the micturition reflex and induced overflow incontinence in five out of 10 animals. Baclofen slightly increased ICI and BC and reduced MF. Conclusion and Implications Our findings demonstrate, for the first time, that a GABAB PAM has potential as a novel approach for the treatment of OAB. PMID:24224799

  16. 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.

  17. 52 Gbps PAM4 receiver sensitivity study for 400GBase-LR8 system using directly modulated laser.

    PubMed

    Motaghiannezam, Reza; Pham, Thang; Chen, Alan; Du, Tengda; Kocot, Chris; Xu, Jack; Huebner, Bernd

    2016-04-01

    Real-time 52 Gbps PAM4 transmission is demonstrated over single mode fiber (SMF) using a directly modulated laser (DML) and a PHY chip. The inner eye optical modulation amplitude (OMA) receiver sensitivities were measured and compared using avalanche photodetector (APD) and PIN photodetector (PD) for the maximum and minimum chromatic dispersions (CDs) of 400GBase-LR8 link. The measured inner eye OMAs were -17.8 dBm and -18.8 dBm for + 10 ps/nm and -58 ps/nm of CDs at the KP4 bit error rate (BER) threshold of 2 × 10-4 using a PIN PD, respectively. The measured inner eye OMA was improved to -21.0 dBm for -58 ps/nm of CD at the KP4 BER threshold using an APD. Negligible OMA penalty (< 0.4 dB) was captured for operating DML at different bias currents of 40 mA and 60 mA using a PIN PD and an APD for both positive and negative CDs at the KP4 BER threshold. PMID:27137027

  18. 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

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

    PubMed

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

    2016-09-20

    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 [(3)H]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

  20. Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

    PubMed

    Dean, Brian; Hopper, Shaun; Conn, P Jeffrey; Scarr, Elizabeth

    2016-05-01

    Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

  1. Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

    PubMed

    DeHaven-Hudkins, D L; Ford-Rice, F Y; Allen, J T; Hudkins, R L

    1993-01-01

    The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo. PMID:8515681

  2. mGlu2 Receptor Agonism, but Not Positive Allosteric Modulation, Elicits Rapid Tolerance towards Their Primary Efficacy on Sleep Measures in Rats.

    PubMed

    Ahnaou, Abdallah; Lavreysen, Hilde; Tresadern, Gary; Cid, Jose M; Drinkenburg, Wilhelmus H

    2015-01-01

    G-protein-coupled receptor (GPCR) agonists are known to induce both cellular adaptations resulting in tolerance to therapeutic effects and withdrawal symptoms upon treatment discontinuation. Glutamate neurotransmission is an integral part of sleep-wake mechanisms, which processes have translational relevance for central activity and target engagement. Here, we investigated the efficacy and tolerance potential of the metabotropic glutamate receptors (mGluR2/3) agonist LY354740 versus mGluR2 positive allosteric modulator (PAM) JNJ-42153605 on sleep-wake organisation in rats. In vitro, the selectivity and potency of JNJ-42153605 were characterized. In vivo, effects on sleep measures were investigated in rats after once daily oral repeated treatment for 7 days, withdrawal and consecutive re-administration of LY354740 (1-10 mg/kg) and JNJ-42153605 (3-30 mg/kg). JNJ-42153605 showed high affinity, potency and selectivity at mGluR2. Binding site analyses and knowledge-based docking confirmed the specificity of JNJ-42153605 at the mGluR2 allosteric binding site. Acute LY354740 and JNJ-42153605 dose-dependently decreased rapid eye movement (REM) sleep time and prolonged its onset latency. Sub chronic effects of LY354740 on REM sleep measures disappeared from day 3 onwards, whereas those of JNJ-42153605 were maintained after repeated exposure. LY354740 attenuated REM sleep homeostatic recovery, while this was preserved after JNJ-42153605 administration. JNJ-42153605 enhanced sleep continuity and efficiency, suggesting its potential as an add-on medication for impaired sleep quality during early stages of treatment. Abrupt cessation of JNJ-42153605 did not induce withdrawal phenomena and sleep disturbances, while the initial drug effect was fully reinstated after re-administration. Collectively, long-term treatment with JNJ-42153605 did not induce tolerance phenomena to its primary functional effects on sleep measures, nor adverse effects at withdrawal, while it promoted

  3. mGlu2 Receptor Agonism, but Not Positive Allosteric Modulation, Elicits Rapid Tolerance towards Their Primary Efficacy on Sleep Measures in Rats

    PubMed Central

    Ahnaou, Abdallah; Lavreysen, Hilde; Tresadern, Gary; Cid, Jose M.; Drinkenburg, Wilhelmus H.

    2015-01-01

    G-protein-coupled receptor (GPCR) agonists are known to induce both cellular adaptations resulting in tolerance to therapeutic effects and withdrawal symptoms upon treatment discontinuation. Glutamate neurotransmission is an integral part of sleep-wake mechanisms, which processes have translational relevance for central activity and target engagement. Here, we investigated the efficacy and tolerance potential of the metabotropic glutamate receptors (mGluR2/3) agonist LY354740 versus mGluR2 positive allosteric modulator (PAM) JNJ-42153605 on sleep-wake organisation in rats. In vitro, the selectivity and potency of JNJ-42153605 were characterized. In vivo, effects on sleep measures were investigated in rats after once daily oral repeated treatment for 7 days, withdrawal and consecutive re-administration of LY354740 (1–10 mg/kg) and JNJ-42153605 (3–30 mg/kg). JNJ-42153605 showed high affinity, potency and selectivity at mGluR2. Binding site analyses and knowledge-based docking confirmed the specificity of JNJ-42153605 at the mGluR2 allosteric binding site. Acute LY354740 and JNJ-42153605 dose-dependently decreased rapid eye movement (REM) sleep time and prolonged its onset latency. Sub chronic effects of LY354740 on REM sleep measures disappeared from day 3 onwards, whereas those of JNJ-42153605 were maintained after repeated exposure. LY354740 attenuated REM sleep homeostatic recovery, while this was preserved after JNJ-42153605 administration. JNJ-42153605 enhanced sleep continuity and efficiency, suggesting its potential as an add-on medication for impaired sleep quality during early stages of treatment. Abrupt cessation of JNJ-42153605 did not induce withdrawal phenomena and sleep disturbances, while the initial drug effect was fully reinstated after re-administration. Collectively, long-term treatment with JNJ-42153605 did not induce tolerance phenomena to its primary functional effects on sleep measures, nor adverse effects at withdrawal, while it promoted

  4. 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.

  5. 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.

  6. 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. PMID:27007499

  7. 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

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

    PubMed

    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

    2015-09-24

    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

  9. 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

  10. Synthesis and biological evaluation of indole-2-carboxamides bearing photoactivatable functionalities as novel allosteric modulators for the cannabinoid CB1 receptor.

    PubMed

    Qiao, Chang-Jiang; Ali, Hamed I; Ahn, Kwang H; Kolluru, Srikanth; Kendall, Debra A; Lu, Dai

    2016-10-01

    5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569, 1) is a prototypical allosteric modulator for the cannabinoid CB1 receptor. Based on this indole-2-carboxamide scaffold, we designed and synthesized novel CB1 allosteric modulators that possess photoactivatable functionalities, which include benzophenone, phenyl azide, aliphatic azide and phenyltrifluoromethyldiazrine. To assess their allosteric effects, the dissociation constant (KB) and allosteric binding cooperativity factor (α) were determined and compared to their parent compounds. Within this series, benzophenone-containing compounds 26 and 27, phenylazide-containing compound 28, and the aliphatic azide containing compound 36b showed allosteric binding parameters (KB and α) comparable to their parent compound 1, 7, 8, and 9, respectively. We further assessed these modulators for their impact on G-protein coupling activity. Interestingly, these compounds exhibited negative allosteric modulator properties in a manner similar to their parent compounds, which antagonize agonist-induced G-protein coupling. These novel CB1 allosteric modulators, possessing photoactivatable functionalities, provide valuable tools for future photo-affinity labeling and mapping the CB1 allosteric binding site(s). PMID:27318976

  11. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

  12. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins. PMID:27526031

  13. Imidazenil, a partial positive allosteric modulator of GABAA receptors, exhibits low tolerance and dependence liabilities in the rat.

    PubMed

    Auta, J; Giusti, P; Guidotti, A; Costa, E

    1994-09-01

    Long-term treatment of rats with full (triazolam) or selective (diazepam) allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors rapidly induced tolerance to the protective effect of these drugs against bicuculline-induced convulsion. In contrast, long-term administration of partial allosteric modulators (imidazenil and bretazenil) of GABAA receptors, in doses equipotent to those of diazepam and triazolam that induce anticonvulsant tolerance, failed to elicit such a tolerance. Furthermore, no cross-tolerance was observed between diazepam and imidazenil. Discontinuation of long-term treatment with diazepam or triazolam, but not of long-term treatment with imidazenil or bretazenil, sensitized rats to behavioral inhibition by punishment (electric shock) in a manner that was potentiated by flumazenil. Administration of a single oral dose of [14C]diazepam or [3H] imidazenil to rats treated repeatedly with the corresponding unlabeled drug or vehicle revealed that the brain concentrations of drugs and their metabolites were similar in both groups of animals. This suggests that tolerance to the full or selective allosteric modulators of GABAA receptors may be associated with changes in the efficacy of the allosteric modulation rather than with changes in drug metabolism. Imidazenil has a longer half-life than an equipotent dose of diazepam and protects rats against bicuculline-induced convulsions for a significantly longer time than diazepam or bretazenil.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Pharmacological stimulation of metabotropic glutamate receptor type 4 in a rat model of Parkinson's disease and l-DOPA-induced dyskinesia: Comparison between a positive allosteric modulator and an orthosteric agonist

    PubMed Central

    Iderberg, Hanna; Maslava, Natallia; Thompson, Analisa D.; Bubser, Michael; Niswender, Colleen M.; Hopkins, Corey R.; Lindsley, Craig W.; Conn, P. Jeffrey; Jones, Carrie K.; Cenci, M. Angela

    2015-01-01

    Metabotropic glutamate receptor 4 (mGlu4) negatively modulates GABA and glutamate release in the ‘indirect pathway’ of the basal ganglia, and has thus been proposed as a potential target to treat motor symptoms in Parkinson's disease. Here, we present an extensive comparison of the behavioural effects produced by the mGlu4 positive allosteric modulator (PAM), VU0364770, and the mGlu4 orthosteric agonist, LSP1-2111, in rats with unilateral 6-OHDA lesions. The compounds' activity was initially assessed in a test of haloperidol-induced catalepsy in intact rats, and effective doses were then evaluated in the hemiparkinsonian animal model. Neither of the two compounds modified the development of dyskinetic behaviours elicited by chronic treatment with full doses of l-DOPA. When given together with l-DOPA to rats with already established dyskinesias, neither VU0364770 nor LSP1-2111 modified the abnormal involuntary movement scores. VU0364770 potentiated, however, the motor stimulant effect of a sub-threshold l-DOPA dose in certain behavioural tests, whereas LSP1-2111 lacked this ability. Taken together, these results indicate that a pharmacological stimulation of mGlu4 lacks intrinsic antidyskinetic activity, but may have DOPA-sparing activity in Parkinson's disease. For the latter indication, mGlu4 PAMs appear to provide a better option than orthosteric agonists. PMID:25749357

  15. Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Reveals Chemical and Functional Diversity and In Vivo Activity in Rat Behavioral Models of Anxiolytic and Antipsychotic ActivityS⃞

    PubMed Central

    Rodriguez, Alice L.; Grier, Mark D.; Jones, Carrie K.; Herman, Elizabeth J.; Kane, Alexander S.; Smith, Randy L.; Williams, Richard; Zhou, Ya; Marlo, Joy E.; Days, Emily L.; Blatt, Tasha N.; Jadhav, Satyawan; Menon, Usha N.; Vinson, Paige N.; Rook, Jerri M.; Stauffer, Shaun R.; Niswender, Colleen M.; Lindsley, Craig W.; Weaver, C. David

    2010-01-01

    Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have

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

    PubMed

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

    2011-02-15

    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 α(2A)-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 G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) 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

  17. Modulation of Calmodulin Lobes by Different Targets: An Allosteric Model with Hemiconcerted Conformational Transitions

    PubMed Central

    Lai, Massimo; Brun, Denis; Edelstein, Stuart J.; Le Novère, Nicolas

    2015-01-01

    Calmodulin is a calcium-binding protein ubiquitous in eukaryotic cells, involved in numerous calcium-regulated biological phenomena, such as synaptic plasticity, muscle contraction, cell cycle, and circadian rhythms. It exibits a characteristic dumbell shape, with two globular domains (N- and C-terminal lobe) joined by a linker region. Each lobe can take alternative conformations, affected by the binding of calcium and target proteins. Calmodulin displays considerable functional flexibility due to its capability to bind different targets, often in a tissue-specific fashion. In various specific physiological environments (e.g. skeletal muscle, neuron dendritic spines) several targets compete for the same calmodulin pool, regulating its availability and affinity for calcium. In this work, we sought to understand the general principles underlying calmodulin modulation by different target proteins, and to account for simultaneous effects of multiple competing targets, thus enabling a more realistic simulation of calmodulin-dependent pathways. We built a mechanistic allosteric model of calmodulin, based on an hemiconcerted framework: each calmodulin lobe can exist in two conformations in thermodynamic equilibrium, with different affinities for calcium and different affinities for each target. Each lobe was allowed to switch conformation on its own. The model was parameterised and validated against experimental data from the literature. In spite of its simplicity, a two-state allosteric model was able to satisfactorily represent several sets of experiments, in particular the binding of calcium on intact and truncated calmodulin and the effect of different skMLCK peptides on calmodulin’s saturation curve. The model can also be readily extended to include multiple targets. We show that some targets stabilise the low calcium affinity T state while others stabilise the high affinity R state. Most of the effects produced by calmodulin targets can be explained as modulation

  18. 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.

  19. Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.

    PubMed

    Rodriguez, Hector M; Vaysberg, Maria; Mikels, Amanda; McCauley, Scott; Velayo, Arleene C; Garcia, Carlos; Smith, Victoria

    2010-07-01

    In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

  20. Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins.

    PubMed

    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

    2011-05-01

    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 Thr(342) of the reactive loop and Tyr(241) 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 Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). 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.

  1. Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

    PubMed Central

    2012-01-01

    Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

  2. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  3. 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

    2014-10-01

    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.

  4. 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

  5. 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

  6. Structure-activity relationships of substituted 1H-indole-2-carboxamides as CB1 receptor allosteric modulators.

    PubMed

    Nguyen, Thuy; German, Nadezhda; Decker, Ann M; Li, Jun-Xu; Wiley, Jenny L; Thomas, Brian F; Kenakin, Terry P; Zhang, Yanan

    2015-05-01

    A series of substituted 1H-indole-2-carboxamides structurally related to compounds Org27569 (1), Org29647 (2) and Org27759 (3) were synthesized and evaluated for CB1 allosteric modulating activity in calcium mobilization assays. Structure-activity relationship studies showed that the modulation potency of this series at the CB1 receptor was enhanced by the presence of a diethylamino group at the 4-position of the phenyl ring, a chloro or fluoro group at the C5 position and short alkyl groups at the C3 position on the indole ring. The most potent compound (45) had an IC₅₀ value of 79 nM which is ∼2.5 and 10 fold more potent than the parent compounds 3 and 1, respectively. These compounds appeared to be negative allosteric modulators at the CB1 receptor and dose-dependently reduced the Emax of agonist CP55,940. These analogs may provide the basis for further optimization and use of CB1 allosteric modulators.

  7. Modulation of Global Low-Frequency Motions Underlies Allosteric Regulation: Demonstration in CRP/FNR Family Transcription Factors

    PubMed Central

    Burnell, David; Jones, Matthew L.; Richards, Shane A.; McLeish, Tom C. B.; Pohl, Ehmke; Wilson, Mark R.; Cann, Martin J.

    2013-01-01

    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

  8. 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

  9. Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation.

    PubMed

    Hans, M; Wilhelm, M; Swandulla, D

    2012-06-01

    In this study, we have investigated how the function of native and recombinant nicotinic acetylcholine receptors (nAChRs) is modulated by the monoterpenoid alcohol from peppermint (-) menthol. In trigeminal neurons (TG), we found that nicotine (75 μM)-activated whole-cell currents through nAChRs were reversibly reduced by menthol in a concentration-dependent manner with an IC₅₀ of 111 μM. To analyze the mechanism underlying menthol's action in more detail, we used single channel and whole-cell recordings from recombinant human α4β2 nAChR expressed in HEK tsA201 cells. Here, we found a shortening of channel open time and a prolongation of channel closed time, and an increase in single channel amplitude leading in summary to a reduction in single channel current. Furthermore, menthol did not affect nicotine's EC₅₀ value for currents through recombinant human α4β2 nAChRs but caused a significant reduction in nicotine's efficacy. Taken together, these findings indicate that menthol is a negative allosteric modulator of nAChRs.

  10. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

    PubMed Central

    Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  11. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities.

    PubMed

    Hubbard, Paul A; Moody, Colleen L; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  12. Iterative experimental and virtual high-throughput screening identifies metabotropic glutamate receptor subtype 4 positive allosteric modulators

    PubMed Central

    Mueller, Ralf; Dawson, Eric S.; Niswender, Colleen M.; Butkiewicz, Mariusz; Hopkins, Corey R.; Weaver, C. David; Lindsley, Craig W.; Conn, P. Jeffrey; Meiler, Jens

    2013-01-01

    Activation of metabotropic glutamate receptor subtype 4 has been shown to be efficacious in rodent models of Parkinson’s disease. Artificial neural networks were trained based on a recently reported high throughput screen which identified 434 positive allosteric modulators of metabotropic glutamate receptor subtype 4 out of a set of approximately 155,000 compounds. A jury system containing three artificial neural networks achieved a theoretical enrichment of 15.4 when selecting the top 2% compounds of an independent test dataset. The model was used to screen an external commercial database of approximately 450,000 drug-like compounds. 1,100 predicted active small molecules were tested experimentally using two distinct assays of mGlu4 activity. This experiment yielded 67 positive allosteric modulators of metabotropic glutamate receptor subtype 4 that confirmed in both experimental systems. Compared to the 0.3% active compounds in the primary screen, this constituted an enrichment of 22 fold. PMID:22592386

  13. 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

  14. Novel 2- and 4- Substituted 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as Allosteric Modulators of the A3 Adenosine Receptor

    PubMed Central

    Kim, Yoonkyung; de Castro, Sonia; Gao, Zhan-Guo; IJzerman, Adriaan P.; Jacobson, Kenneth A.

    2009-01-01

    4-Arylamino and 2- cycloalkyl (including amino substitution) modifications were made in a series of 1H-imidazo-[4,5-c]quinolin-4-amine derivatives as allosteric modulators of the human A3 adenosine receptor (AR). In addition to allosteric modulation of the maximum functional efficacy (in [35S]GTPγS G protein binding assay) of the A3AR agonist Cl-IB-MECA (15), some analogues also weakly inhibited equilibrium radioligand binding at ARs. 4-(3,5-Dichlorophenylamino) (6) or 2-(1-adamantyl) (20) substitution produced allosteric enhancement (twice the maximal agonist efficacy), with minimal inhibition of orthosteric AR binding. 2-(4-Tetrahydropyranyl) substitution abolished allosteric enhancement but preserved inhibition of orthosteric binding. Introduction of nitrogen in the six-membered ring at 2 position, to improve aqueous solubility and provide a derivatization site, greatly reduced the allosteric enhancement. 2-(4-(Benzoylamino)cyclohexyl) analogues 23 and 24 were weak negative A3AR modulators. Thus, consistent with previous findings, the allosteric and orthosteric inhibitory A3AR effects in imidazoquinolines are structurally separable, suggesting the possible design of additional derivatives with enhanced positive or negative allosteric A3AR activity and improved selectivity in comparison to inhibition of orthosteric binding. PMID:19284749

  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. 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

  17. Global allostery model of hemoglobin. Modulation of O(2) affinity, cooperativity, and Bohr effect by heterotropic allosteric effectors.

    PubMed

    Yonetani, Takashi; Park, Sung-Ick; Tsuneshige, Antonio; Imai, Kiyohiro; Kanaori, Kenji

    2002-09-13

    The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.

  18. Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator.

    PubMed

    Hamouda, Ayman K; Deba, Farah; Wang, Ze-Jun; Cohen, Jonathan B

    2016-05-01

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing theα4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [(3)H]CMPI upon photolysis at 312 nm to identify its binding sites inTorpedonAChRs. Recording fromXenopusoocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2nAChR to 10μM ACh (EC10) by 400% and with anEC50of ∼1µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10µM) ACh responses of (α4)2(β2)3nAChRs and fully inhibited human muscle andTorpedonAChRs with IC50values of ∼0.5µM. Upon irradiation at 312 nm, [(3)H]CMPI photoincorporated into eachTorpedo[(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [(3)H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr(190),αTyr(198),γTrp(55),γTyr(111),γTyr(117),δTrp(57)) that was fully inhibitable by agonist and lower-efficiency, state-dependent [(3)H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing anα4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2nAChR. PMID:26976945

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

    PubMed

    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.

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

    PubMed

    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

  2. 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

  3. Effect of the novel positive allosteric modulator of mGluR2 AZD8529 on incubation of methamphetamine craving after prolonged voluntary abstinence in a rat model

    PubMed Central

    Caprioli, Daniele; Venniro, Marco; Zeric, Tamara; Li, Xuan; Adhikary, Sweta; Madangopal, Rajtarun; Marchant, Nathan J.; Lucantonio, Federica; Schoenbaum, Geoffrey; Bossert, Jennifer M.; Shaham, Yavin

    2015-01-01

    Background Cue-induced methamphetamine craving increases after prolonged forced (experimenter-imposed) abstinence from the drug (incubation of methamphetamine craving). Here, we determined whether this incubation phenomenon would occur under conditions that promote voluntary (self-imposed) abstinence. We also determined the effect of the novel mGluR2 positive allosteric modulator, AZD8529, on incubation of methamphetamine craving after forced or voluntary abstinence. Methods We trained rats to self-administer palatable food (6 sessions) and then to self-administer methamphetamine under two conditions: 12 sessions (9-hr/day) or 50 sessions (3-hr/day). We then assessed cue-induced methamphetamine seeking in extinctions test after 1 or 21 abstinence days. Between tests, the rats underwent either forced abstinence (no access to the food- or drug-paired levers) or voluntary abstinence for 19 days (achieved via a discrete choice procedure between methamphetamine and palatable food; 20 trials per day). We also determined the effect of subcutaneous injections of AZD8529 (20 and 40 mg/kg) on cue-induced methamphetamine seeking 1 or 21 days after forced or voluntary abstinence. Results Under both training and abstinence conditions, cue-induced methamphetamine seeking in the extinction tests was higher after 21 abstinence days than after 1 day (incubation of methamphetamine craving). AZD8529 decreased cue-induced methamphetamine seeking on day 21 but not day 1 of forced or voluntary abstinence. Conclusions We introduce a novel animal model to study incubation of drug craving and cue-induced drug seeking after prolonged voluntary abstinence, mimicking the human condition of relapse after successful contingency management treatment. Our data suggest that PAMs of mGluR2 should be considered for relapse prevention. PMID:25861699

  4. In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569.

    PubMed

    Gamage, Thomas F; Ignatowska-Jankowska, Bogna M; Wiley, Jenny L; Abdelrahman, Mostafa; Trembleau, Laurent; Greig, Iain R; Thakur, Ganesh A; Tichkule, Ritesh; Poklis, Justin; Ross, Ruth A; Pertwee, Roger G; Lichtman, Aron H

    2014-04-01

    Several allosteric modulators (AMs) of the CB1 receptor have been characterized in vitro, including Org27569, which enhances CB1-specific binding of [H]CP55,940, but behaves as an insurmountable CB1-receptor antagonist in several biochemical assays. Although a growing body of research has investigated the molecular actions of this unusual AM, it is unknown whether these actions translate to the whole animal. The purpose of the present study was to determine whether Org27569 would produce effects in well-established mouse behavioral assays sensitive to CB1 orthosteric agonists and antagonists. Similar to the orthosteric CB1 antagonist/inverse agonist rimonabant, Org27569 reduced food intake; however, this anorectic effect occurred independently of the CB1 receptor. Org27569 did not elicit CB1-mediated effects alone and lacked efficacy in altering antinociceptive, cataleptic, and hypothermic actions of the orthosteric agonists anandamide, CP55,940, and Δ-tetrahydrocannabinol. Moreover, it did not alter the discriminative stimulus effects of anandamide in FAAH-deficient mice or Δ-tetrahydrocannabinol in wild-type mice in the drug discrimination paradigm. These findings question the utility of Org27569 as a 'gold standard' CB1 AM and underscore the need for the development of CB1 AMs with pharmacology that translates from the molecular level to the whole animal. PMID:24603340

  5. Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor.

    PubMed

    Ahn, Kwang H; Mahmoud, Mariam M; Samala, Sushma; Lu, Dai; Kendall, Debra A

    2013-03-01

    Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced β-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

  6. Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).

    PubMed

    Maki, Nazli; Dey, Saibal

    2006-07-14

    The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

  7. PAM stack test utility

    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.

  8. Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors

    PubMed Central

    Nörenberg, W; Sobottka, H; Hempel, C; Plötz, T; Fischer, W; Schmalzing, G; Schaefer, M

    2012-01-01

    BACKGROUND AND PURPOSE In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca2+]i influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG+ permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues. PMID:22506590

  9. 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

  10. Positive allosteric modulators of the human sweet taste receptor enhance sweet taste.

    PubMed

    Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E; Zoller, Mark; Karanewsky, Donald S

    2010-03-01

    To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 microM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 microM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 microM) and SE-3 (200 microM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 microM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 microM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste. PMID:20173092

  11. Positive allosteric modulators of the human sweet taste receptor enhance sweet taste

    PubMed Central

    Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E.; Zoller, Mark; Karanewsky, Donald S.

    2010-01-01

    To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste. PMID:20173092

  12. Positive allosteric modulators of the human sweet taste receptor enhance sweet taste.

    PubMed

    Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E; Zoller, Mark; Karanewsky, Donald S

    2010-03-01

    To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 microM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 microM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 microM) and SE-3 (200 microM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 microM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 microM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.

  13. Block and allosteric modulation of GABAergic currents by oenanthotoxin in rat cultured hippocampal neurons

    PubMed Central

    Wyrembek, Paulina; Lebida, Katarzyna; Mercik, Katarzyna; Szczuraszek, Katarzyna; Szczot, Marcin; Pollastro, Federica; Appendino, Giovanni; Mozrzymas, Jerzy W

    2010-01-01

    Background and purpose: Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABAA receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX. Experimental approach: Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches. Key results: OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABAA receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs. Conclusions and implications: OETX shows a complex blocking mechanism of GABAA receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX. PMID:20590622

  14. 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

  15. Effects of negative allosteric modulators of gamma-aminobutyric acidA receptors on complex behavioral processes in monkeys.

    PubMed

    Auta, J; Winsauer, P J; Faust, W B; Lambert, P; Moerschbaecher, J M

    1997-01-01

    A multiple schedule of repeated acquisition and performance of conditional discriminations was used to characterize the effects of two negative allosteric modulators of the gamma-aminobutyric acid (GABAA) receptor (ethyl beta-carboline-3-carboxylate [beta-CCE] and N-methyl-beta-carboline-3-carboxamide [FG-7142]), a hallucinogenic beta-carboline derivative (harmine), a benzodiazepine receptor antagonist (flumazenil) and a positive allosteric modulator (alprazolam). In the acquisition component, subjects acquired a different discrimination each session. Acquisition of a discrimination was defined by a decrease in errors as the session progressed. In the performance component, the discrimination was the same each session. Responding in both components was maintained by food presentation under a variable-ratio schedule. Incorrect responses in both components produced a 5-sec timeout. Alprazolam (0.1-18 mg/kg), beta-CCE (0.01-0.32 mg/kg), FG-7142 (0.1-18 mg/kg) and harmine (0.1-1.8 mg/kg) all dose-dependently decreased response rate in both components. However, accuracy of responding-was differentially affected by the drugs. Alprazolam selectively and dose-dependently increased percent errors in acquisition, whereas beta-CCE increased acquisition errors only at the highest doses tested in each subject. In contrast, FG-7142 and harmine had no effects on percent errors at doses that virtually eliminated responding. In all cases, performance accuracy was generally not affected. Flumazenil, at doses that had little or no effect (0.1 and 0.32 mg/kg) or occasionally decreased response rates (1 mg/kg) when administered alone, dose-dependently antagonized the rate-decreasing and error-increasing effects of beta-CCE, FG-7142 and alprazolam. In contrast, flumazenil failed to antagonize the effects of harmine. Thus, the negative allosteric modulators only moderately disrupted acquisition in comparison with the positive allosteric modulator, but the effects of both types of

  16. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: Lead optimization.

    PubMed

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kolok, Sándor; Mikó-Bakk, Mónika L; Vastag, Mónika; Sághy, Katalin; Gyertyán, István; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2015-04-15

    An HTS campaign of our corporate compound library, and hit-to lead development resulted in thieno[2,3-b]pyridine derivative leads with mGluR5 negative allosteric modulator effects. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modification of the first two targeted regions resulted in compounds with nanomolar affinity, then optimal substitution of the third region improved metabolic stability. One of the most promising compounds showed excellent in vivo efficacy and is a potential development candidate.

  17. The role of the M4 lipid-sensor in the folding, trafficking, and allosteric modulation of nicotinic acetylcholine receptors.

    PubMed

    Hénault, Camille M; Sun, Jiayin; Therien, J P Daniel; daCosta, Corrie J B; Carswell, Casey L; Labriola, Jonathan M; Juranka, Peter F; Baenziger, John E

    2015-09-01

    With the availability of high resolution structural data, increasing attention has focused on the mechanisms by which drugs and endogenous compounds allosterically modulate nicotinic acetylcholine receptor (nAChR) function. Lipids are potent modulators of the nAChR from Torpedo. Membrane lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations, as well as influencing the transitions between these conformational states. Structural and functional data highlight a role for the lipid-exposed M4 transmembrane α-helix of each subunit in lipid sensing, and suggest that lipids influence gating by altering the binding of M4 to the adjacent transmembrane α-helices, M1 and M3. M4 has also been implicated in both the folding and trafficking of nAChRs to the cell surface, as well as in the potentiation of nAChR gating by neurosteroids. Here, we discuss the roles of M4 in the folding, trafficking, and allosteric modulation of nAChRs. We also consider the hypothesis that variable chemistry at the M4-M1/M3 transmembrane α-helical interface in different nAChR subunits governs the capacity for potentiation by activating lipids. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25433148

  18. In vitro and in vivo characterization of the novel GABAB receptor positive allosteric modulator, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE).

    PubMed

    Perdona', Elisabetta; Costantini, Vivian J A; Tessari, Michela; Martinelli, Prisca; Carignani, Corrado; Valerio, Enzo; Mok, M H Selina; Zonzini, Laura; Visentini, Filippo; Gianotti, Massimo; Gordon, Laurie; Rocheville, Magalie; Corsi, Mauro; Capelli, Anna Maria

    2011-01-01

    There is preclinical evidence supporting the finding that the GABA(B) receptor orthosteric agonist, baclofen, has significant effects on eating behavior suggesting the potential therapeutic application of this compound for the treatment of eating related disorders. However, the wide clinical use of baclofen might be limited by the appearance of sedative and motor impairment effects. The identification of positive allosteric modulators (PAMs) of GABA(B) receptors represents a novel therapeutic approach to reduce the centrally-mediated adverse effects typical of the GABA(B) receptor orthosteric agonist. In the present work, we report the in vitro profile of a novel chemical structure, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE) identified by screening the GSK compound collection. CMPPE potentiates GABA-stimulated [(35)S]GTPγS binding to membranes of human recombinant cell line and of rat brain cortex. GABA concentration-response curves (CRC) in the presence of fixed concentrations of CMPPE, in rat native tissue, revealed an increase of both the potency and maximal efficacy of GABA. A similar modulatory effect was observed in GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in hippocampal neurons. CMPPE (30-100 mg/kg) and GS39783 (100 mg/kg) significantly decreased food consumption in rat without impairment on the animal locomotor activity. On the contrary, baclofen (2.5 mg/kg) decreased both food intake and motor performance. All together these findings confirm the role of GABA(B) system in controlling animal food intake and for the first time demonstrate that GABA(B) receptor PAMs may represent a novel pharmacological approach to treat eating disorders without unwanted sedative effects.

  19. Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

    PubMed

    Jang, Jae Wan; Cho, Nam-Chul; Min, Sun-Joon; Cho, Yong Seo; Park, Ki Duk; Seo, Seon Hee; No, Kyoung Tai; Pae, Ae Nim

    2016-02-01

    Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators.

  20. Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

    PubMed

    Jang, Jae Wan; Cho, Nam-Chul; Min, Sun-Joon; Cho, Yong Seo; Park, Ki Duk; Seo, Seon Hee; No, Kyoung Tai; Pae, Ae Nim

    2016-02-01

    Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators. PMID:26343933

  1. The M1 Muscarinic Positive Allosteric Modulator PQCA Improves Performance on Translatable Tests of Memory and Attention in Rhesus Monkeys.

    PubMed

    Lange, Henry S; Cannon, Christopher E; Drott, Jason T; Kuduk, Scott D; Uslaner, Jason M

    2015-12-01

    Improved treatment of Alzheimer disease (AD) is a significant unmet medical need that is becoming even more critical given the rise in the number of patients and the substantial economic burden. The current standards of care, acetylcholinesterase inhibitors (AChEIs), are hindered by gastrointestinal side effects owing to their nonselective activation of muscarinic and nicotinic receptors. Recently, the highly selective M1 positive allosteric modulator PQCA (1-((4-cyano-4-(pyridine-2-yl)piperidin-1-yl)methyl-4-oxo-4 H-quinolizine-3-carboxylic acid) has been demonstrated to improve cognition in a variety of rodent and nonhuman primate cognition models without producing significant gastrointestinal side effects. Here we describe the effect of PQCA and the AChEI donepezil on two clinically relevant and highly translatable touchscreen cognition tasks in nonhuman primates: paired-associates learning (PAL) and the continuous-performance task (CPT). Blockade of muscarinic signaling by scopolamine produced significant impairments in both PAL and CPT. PQCA and donepezil attenuated the scopolamine deficits in both tasks, and the action of these two compounds was similar in magnitude. In addition, the combination of subeffective doses of PQCA and donepezil enhanced PAL performance. These results further suggest that M1-positive allosteric modulators, either as monotherapy or as an add-on to current standards of care, have potential to reduce the cognitive deficits associated with AD. PMID:26446308

  2. 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

  3. 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). PMID:27318559

  4. 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.

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

    PubMed

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

    2015-10-20

    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.

  6. 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.

  7. 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

  8. The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator.

    PubMed

    Fisher, Janet L

    2009-01-01

    Stiripentol (STP) has been used as co-therapy for treatment of epilepsy for many years. Its mechanism of action has long been considered to be indirect, as it inhibits the enzymes responsible for metabolism of other anti-convulsant agents. However, a recent report suggested that STP might also act at the neuronal level, increasing inhibitory GABAergic neurotransmission. We examined the effect of STP on the functional properties of recombinant GABA(A) receptors (GABARs) and found that it was a positive allosteric modulator of these ion channels. Its activity showed some dependence on subunit composition, with greater potentiation of alpha3-containing receptors and reduced potentiation when the beta1 or epsilon subunits were present. STP caused a leftward shift in the GABA concentration-response relationship, but did not increase the peak response of the receptors to a maximal GABA concentration. Although STP shares some functional characteristics with the neurosteroids, its activity was not inhibited by a neurosteroid site antagonist and was unaffected by a mutation in the alpha3 subunit that reduced positive modulation by neurosteroids. The differential effect of STP on beta1- and beta2/beta3-containing receptors was not altered by mutations within the second transmembrane domain that affect modulation by loreclezole. These findings suggest that STP acts as a direct allosteric modulator of the GABAR at a site distinct from many commonly used anti-convulsant, sedative and anxiolytic drugs. Its higher activity at alpha3-containing receptors as well as its activity at delta-containing receptors may provide a unique opportunity to target selected populations of GABARs.

  9. The mGluR2 Positive Allosteric Modulator BINA Decreases Cocaine Self-Administration and Cue-Induced Cocaine-Seeking and Counteracts Cocaine-Induced Enhancement of Brain Reward Function in Rats

    PubMed Central

    Jin, Xinchun; Semenova, Svetlana; Yang, Li; Ardecky, Robert; Sheffler, Douglas J; Dahl, Russell; Conn, P Jeffrey; Cosford, Nicholas DP; Markou, Athina

    2010-01-01

    Metabotropic glutamate receptor 2/3 (mGluR2/3) agonists were shown previously to nonselectively decrease both cocaine- and food-maintained responding in rats. mGluR2 positive allosteric modulators (PAMs) may represent improved therapeutic compounds because of their modulatory properties and higher selectivity for mGluR2. We analyzed the effects of the selective, brain penetrant, and systemically active mGluR2 PAM potassium 3′-([(2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy]methyl)biphenyl l-4-carboxylate (BINA) and the mGluR2/3 agonist LY379268 on intravenous cocaine self-administration and cocaine-seeking behavior in rats that had short (1 h, ShA) or long (6 h, LgA) access to cocaine. The effects of BINA on food responding and food-seeking behavior were also analyzed. Finally, we examined the effects of BINA on brain reward function and cocaine-induced reward enhancement using the intracranial self-stimulation procedure. BINA decreased cocaine self-administration in both ShA and LgA rats, with no effect on food self-administration. Alternatively, LY379268 nonselectively decreased both cocaine and food self-administration. BINA decreased cue-induced reinstatement of cocaine seeking with no effect on food seeking. The cocaine-induced enhancement of brain reward function was blocked by BINA, although the highest doses of BINA decreased brain reward function when administered alone, suggesting additive, rather than interactive, effects of BINA and cocaine. In conclusion, BINA attenuated the reinforcing and counteracted the reward-enhancing effects of cocaine and decreased cue-induced cocaine-seeking behavior, without affecting behaviors motivated by food reinforcement. The higher selectivity of BINA compared with an mGluR2/3 agonist for drug- vs food-motivated behaviors suggests a therapeutic role for mGluR2 PAMs for the treatment of cocaine addiction and possibly other drugs of abuse. PMID:20555310

  10. Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor.

    PubMed

    Abdul-Ridha, Alaa; Lane, J Robert; Mistry, Shailesh N; López, Laura; Sexton, Patrick M; Scammells, Peter J; Christopoulos, Arthur; Canals, Meritxell

    2014-11-28

    Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs. PMID:25326383

  11. Mechanistic Insights into Allosteric Structure-Function Relationships at the M1 Muscarinic Acetylcholine Receptor*

    PubMed Central

    Abdul-Ridha, Alaa; Lane, J. Robert; Mistry, Shailesh N.; López, Laura; Sexton, Patrick M.; Scammells, Peter J.; Christopoulos, Arthur; Canals, Meritxell

    2014-01-01

    Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-4007.35 in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs. PMID:25326383

  12. Positive allosteric modulator of GABAB receptor alters behavioral effects but not afterdischarge progression induced by partial hippocampal kindling.

    PubMed

    Leung, L Stan; Jin, Miao; Chu, Liangwei; Ma, Jingyi

    2016-11-01

    Hippocampal seizures decreased the function of GABAB receptors, which may further increase seizure susceptibility and contribute to development of schizophrenia-like behaviors. Recent literature indicates that GABAB receptor agonist may normalize schizophrenia-like behaviors and prevent drug-induced behavioral sensitization. We hypothesized that positive modulation of GABAB receptor function during seizure induction will reduce seizure-induced schizophrenia-like behaviors. Using a partial hippocampal kindling model, afterdischarges were induced after injection of saline or dimethyl sulfoxide (vehicle-kindled rats), or a GABAB receptor positive allosteric modulator CGP7930, at 1 mg/kg i.p. (CGP1-kindled) or 5 mg/kg i.p. (CGP5-kindled). The increase in the primary afterdischarge duration during kindling was not different among the groups. However, the CGP5-kindled group showed a lower afterdischarge starting frequency as compared to vehicle-kindled or CGP1-kindled groups. Partial hippocampal kindling (21 afterdischarges) resulted in decreased prepulse inhibition and decreased gating of hippocampal auditory evoked potentials in vehicle-kindled and CGP1-kindled rats, as compared to saline-injected non-kindled rats, recorded 3-4 days after the last afterdischarge. However, CGP5-kindled rats showed normal prepulse inhibition and hippocampal auditory gating (compared to non-kindled rats), which was significantly higher than the respective measure in vehicle-kindled rats. CGP5-kindled group also showed methamphetamine-induced locomotion that was significant lower than the vehicle-kindled or CGP1-kindled group, but slightly higher than the saline-injected non-kindled rats. In conclusion, this study provides original data that a GABAB receptor positive allosteric modulator could therapeutically prevent or normalize some seizure-induced behavioral disruptions in a model of temporal lobe epilepsy. PMID:27436721

  13. Positive allosteric modulator of GABAB receptor alters behavioral effects but not afterdischarge progression induced by partial hippocampal kindling.

    PubMed

    Leung, L Stan; Jin, Miao; Chu, Liangwei; Ma, Jingyi

    2016-11-01

    Hippocampal seizures decreased the function of GABAB receptors, which may further increase seizure susceptibility and contribute to development of schizophrenia-like behaviors. Recent literature indicates that GABAB receptor agonist may normalize schizophrenia-like behaviors and prevent drug-induced behavioral sensitization. We hypothesized that positive modulation of GABAB receptor function during seizure induction will reduce seizure-induced schizophrenia-like behaviors. Using a partial hippocampal kindling model, afterdischarges were induced after injection of saline or dimethyl sulfoxide (vehicle-kindled rats), or a GABAB receptor positive allosteric modulator CGP7930, at 1 mg/kg i.p. (CGP1-kindled) or 5 mg/kg i.p. (CGP5-kindled). The increase in the primary afterdischarge duration during kindling was not different among the groups. However, the CGP5-kindled group showed a lower afterdischarge starting frequency as compared to vehicle-kindled or CGP1-kindled groups. Partial hippocampal kindling (21 afterdischarges) resulted in decreased prepulse inhibition and decreased gating of hippocampal auditory evoked potentials in vehicle-kindled and CGP1-kindled rats, as compared to saline-injected non-kindled rats, recorded 3-4 days after the last afterdischarge. However, CGP5-kindled rats showed normal prepulse inhibition and hippocampal auditory gating (compared to non-kindled rats), which was significantly higher than the respective measure in vehicle-kindled rats. CGP5-kindled group also showed methamphetamine-induced locomotion that was significant lower than the vehicle-kindled or CGP1-kindled group, but slightly higher than the saline-injected non-kindled rats. In conclusion, this study provides original data that a GABAB receptor positive allosteric modulator could therapeutically prevent or normalize some seizure-induced behavioral disruptions in a model of temporal lobe epilepsy.

  14. 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

  15. The role of the mGluR allosteric modulation in the NMDA-hypofunction model of schizophrenia.

    PubMed

    Doreulee, N; Alania, M; Mitaishvili, E; Chikovani, M; Chkhartishvili, B

    2009-12-01

    Schizophrenia is one of the most important forms of psychiatric illness and may be chronic and highly disabling. It has been suggested that specific neurochemical abnormality is due to dopaminergic overactivity in the brain. Schizophrenia is currently thought to be associated with a hypoglutamatergic state that is mimicked by acute Phencyclidine (PCP), an antagonist of the N-methyl-D-aspartate (NMDA) receptor subtype. Administration of PCP or ketamine in rodents has been used to model aspects of schizophrenia. Taken into consideration the role of glutamatergic system in development of schizophrenia and involvement of striatal dopaminergic receptors in generation of schizophrenia symptoms, it was planned to study functional interaction between NMDA and metabotropic glutamatergic receptors 5 (mGluR5) in schizophrenia-associated behavioral and memory disturbance and the role of mGluRs allosteric modulation in cortico-striatal synaptic plasticity. In our experiments investigation of dose-dependent effects of ketamine revealed that 0.3mg/kg ketamine induces statistical changes most of behavioral and cognitive parameters in rats. Changes in emotional state showed decrease of the number and total duration of groomings in open field experiments as wall as in passive avoidance task. Decrease of motor activity was also detected, while no significant changes were observed in number of defecations. In T-maze test it was shown that spatial memory was damaged. To determine whether mGlu5 and NMDA receptor interact to regulate complex behaviors that are relevant to cognitive disorders such as schizophrenia we focused on assessing whether the selective mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine MPEP mimics or exacerbates the effects of the NMDA receptor antagonist. Ketamine-induced memory disturbance was significantly increased after injection of mGluR5 negative allosteric modulators MPEP. In In vitro experiments the agonist at group I metabotropic glutamate

  16. DSR-98776, a novel selective mGlu5 receptor negative allosteric modulator with potent antidepressant and antimanic activity.

    PubMed

    Kato, Taro; Takata, Makoto; Kitaichi, Maiko; Kassai, Momoe; Inoue, Mitsuhiro; Ishikawa, Chihiro; Hirose, Wataru; Yoshida, Kozo; Shimizu, Isao

    2015-06-15

    Modulation of monoaminergic systems has been the main stream of treatment for patients with mood disorders. However, recent evidence suggests that the glutamatergic system plays an important role in the pathophysiology of these disorders. This study pharmacologically characterized a structurally novel metabotropic glutamate 5 (mGlu5) receptor negative allosteric modulator, DSR-98776, and evaluated its effect on rodent models of depression and mania. First, DSR-98776 in vitro profile was assessed using intracellular calcium and radioligand binding assays. This compound showed dose-dependent inhibitory activity for mGlu5 receptors by binding to the same allosteric site as 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a known mGlu5 inhibitor. The in vivo therapeutic benefits of DSR-98776 were evaluated in common rodent models of depression and mania. In the rat forced swimming test, DSR-98776 (1-3mg/kg) significantly reduced rats immobility time after treatment for 7 consecutive days, while paroxetine (3 and 10mg/kg) required administration for 2 consecutive weeks to reduce rats immobility time. In the mouse forced swimming test, acute administration of DSR-98776 (10-30 mg/kg) significantly reduced immobility time. This effect was not influenced by 4-chloro-DL-phenylalanine methyl ester hydrochloride-induced 5-HT depletion. Finally, DSR-98776 (30 mg/kg) significantly decreased methamphetamine/chlordiazepoxide-induced hyperactivity in mice, which reflects this compound antimanic-like effect. These results indicate that DSR-98776 acts as an orally potent antidepressant and antimanic in rodent models and can be a promising therapeutic option for the treatment of a broad range of mood disorders with depressive and manic states. PMID:25823809

  17. Antipsychotic profiles of TASP0443294, a novel and orally active positive allosteric modulator of metabotropic glutamate 2 receptor.

    PubMed

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

    2015-03-01

    Glutamatergic dysfunction has been implicated in psychiatric disorders such as schizophrenia. The stimulation of metabotropic glutamate (mGlu) 2 receptor has been shown to be effective in a number of animal models of schizophrenia. In this study, we investigated the antipsychotic profiles of (2S)-5-methyl-2-{[4-(1,1,1-trifluoro-2-methylpropan-2-yl)phenoxy]methyl}-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide (TASP0443294), a newly synthesized positive allosteric modulator of the mGlu2 receptor. TASP0443294 potentiated the response of human mGlu2 and rat mGlu2 receptors to glutamate with EC50 values of 277 and 149 nM, respectively, without affecting the glutamate response of human mGlu3 receptor. TASP0443294 was distributed in the brain and cerebrospinal fluid after peroral administration in rats. The peroral administration of TASP0443294 inhibited methamphetamine-induced hyperlocomotion in rats, which was attenuated by an mGlu2/3 receptor antagonist, and improved social memory impairment induced by 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) in rats. Furthermore, TASP0443294 reduced the ketamine-induced basal gamma hyperactivity in the prefrontal cortex and suppressed rapid eye movement (REM) sleep in rats. These findings indicate that TASP0443294 is an mGlu2 receptor positive allosteric modulator with antipsychotic activity, and that the suppression of aberrant gamma oscillations and REM sleep could be considered as neurophysiological biomarkers for TASP0443294. PMID:25837934

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

    PubMed

    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

    2015-09-18

    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

  19. 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

  20. A selective positive allosteric modulator of metabotropic glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis.

    PubMed

    Benneyworth, Michael A; Xiang, Zixiu; Smith, Randy L; Garcia, Efrain E; Conn, P Jeffrey; Sanders-Bush, Elaine

    2007-08-01

    Recent clinical studies reveal that selective agonists of group II metabotropic glutamate (mGlu) receptors have robust efficacy in treating positive and negative symptoms in patients with schizophrenia. Group II mGlu receptor agonists also modulate the in vivo activity of psychotomimetic drugs and reduce the ability of psychotomimetic hallucinogens to increase glutamatergic transmission. Because increased excitation of the medial prefrontal cortex (mPFC) has been implicated in pathophysiology of schizophrenia, the ability of group II mGlu receptor agonists to reduce hallucinogenic drug action in this region is believed to be directly related to their antipsychotic efficacy. A novel class of ligands, termed positive allosteric modulators, has recently been identified, displaying exceptional mGlu2 receptor selectivity. These compounds do not activate mGlu2 receptors directly but potentiate the ability of glutamate and other agonists to activate this receptor. We now report that the mGlu2 receptor-selective positive allosteric modulator biphenyl-indanone A (BINA) modulates excitatory neurotransmission in the mPFC and attenuates the in vivo actions of the hallucinogenic 5-HT(2A/2C) receptor agonist (-)2,5-dimethoxy-4-bromoamphetamine [(-)DOB]. BINA attenuates serotonin-induced increases in spontaneous excitatory postsynaptic currents in the mPFC, mimicking the effect of the mGlu2/3 receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV). In addition, BINA reduced (-)DOB-induced head twitch behavior and Fos expression in mPFC, effects reversed by pretreatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl) -3 - (xanth-9-yl-)propionic acid (LY341495). These data confirm the relevance of excitatory signaling in the mPFC to the behavioral actions of hallucinogens and further support the targeting of mGlu2 receptors as a novel strategy for treating glutamatergic dysfunction in schizophrenia.

  1. 7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm?

    PubMed

    Citti, Cinzia; Battisti, Umberto M; Cannazza, Giuseppe; Jozwiak, Krzysztof; Stasiak, Natalia; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino; Zoli, Michele

    2016-02-17

    5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1. PMID:26580317

  2. Baclofen and Other GABAB Receptor Agents Are Allosteric Modulators of the CXCL12 Chemokine Receptor CXCR4

    PubMed Central

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

    2013-01-01

    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 Ca2+ 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. PMID:23843532

  3. 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. PMID:27054065

  4. 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

  5. A Novel Class of Succinimide-Derived Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 1 Provides Insight into a Disconnect in Activity between the Rat and Human Receptors

    PubMed Central

    2014-01-01

    Recent progress in the discovery of mGlu1 allosteric modulators has suggested the modulation of mGlu1 could offer possible treatment for a number of central nervous system disorders; however, the available chemotypes are inadequate to fully investigate the therapeutic potential of mGlu1 modulation. To address this issue, we used a fluorescence-based high-throughput screening assay to screen an allosteric modulator-biased library of compounds to generate structurally diverse mGlu1 negative allosteric modulator hits for chemical optimization. Herein, we describe the discovery and characterization of a novel mGlu1 chemotype. This series of succinimide negative allosteric modulators, exemplified by VU0410425, exhibited potent inhibitory activity at rat mGlu1 but was, surprisingly, inactive at human mGlu1. VU0410425 and a set of chemically diverse mGlu1 negative allosteric modulators previously reported in the literature were utilized to examine this species disconnect between rat and human mGlu1 activity. Mutation of the key transmembrane domain residue 757 and functional screening of VU0410425 and the literature compounds suggests that amino acid 757 plays a role in the activity of these compounds, but the contribution of the residue is scaffold specific, ranging from critical to minor. The operational model of allosterism was used to estimate the binding affinities of each compound to compare to functional data. This novel series of mGlu1 negative allosteric modulators provides valuable insight into the pharmacology underlying the disconnect between rat and human mGlu1 activity, an issue that must be understood to progress the therapeutic potential of allosteric modulators of mGlu1. PMID:24798819

  6. 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).

  7. 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). PMID:27410116

  8. 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

  9. Conformational state-dependent anion binding in prestin: evidence for allosteric modulation.

    PubMed

    Song, Lei; Santos-Sacchi, Joseph

    2010-02-01

    Outer hair cells boost auditory performance in mammals. This amplification relies on an expansive array of intramembranous molecular motors, identified as prestin, that drive somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, we are able to assess prestin's conformational state and interrogate the effectiveness of anions on prestin's activity. We find that the affinity of anions depends on the state of prestin that we set with a variety of perturbations (in membrane tension, temperature, and voltage), and that movement into the expanded state reduces the affinity of prestin for anions. These data signify that anions work allosterically on prestin. Consequently, anions are released from prestin's binding site during expansion, i.e., during hyperpolarization. This is at odds with the extrinsic voltage sensor model, which suggests that prestin-bound intracellular anions are propelled deep into the membrane. Furthermore, we hypothesize that prestin's susceptibility to many biophysical forces, and notably its piezoelectric nature, may reflect anion interactions with the motor. PMID:20141749

  10. Tasquinimod is an Allosteric Modulator of HDAC4 Survival Signaling within the Compromised Cancer Microenvironment

    PubMed Central

    Isaacs, John T; Antony, Lizamma; Dalrymple, Susan L; Brennen, W. Nathaniel; Gerber, Stephanie; Hammers, Hans; Wissing, Michel; Kachhap, Sushant; Luo, Jun; Xing, Li; Björk, Per; Olsson, Anders; Björk, Anders; Leanderson, Tomas

    2012-01-01

    Tasquinimod is an orally active anti-angiogenic drug that is currently in Phase III clinical trials for the treatment of castration resistant prostate cancer. However, the target of this drug has remained unclear. In this study we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the anti-angiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10–30 nM) to the regulatory Zn2+ binding domain of HDAC4 which locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited co-localization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. PMID:23149916

  11. Exploration of synthetic approaches and pharmacological evaluation of PNU-69176E and its stereoisomer as 5-HT2C receptor allosteric modulators.

    PubMed

    Ding, Chunyong; Bremer, Nicole M; Smith, Thressa D; Seitz, Patricia K; Anastasio, Noelle C; Cunningham, Kathryn A; Zhou, Jia

    2012-07-18

    Allosteric modulators of the serotonin (5-HT) 5-HT(2C) receptor (5-HT(2C)R) present a unique drug design strategy to augment the response to endogenous 5-HT in a site- and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT(2C)R. For the first time, an optimized synthetic route to readily access PNU-69176E (1) and its diastereomer 2 has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid. This synthetic approach not only enables a feasible preparation of a sufficient amount of 1 for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT(2C)R allosteric modulators. Compound 1 and its diastereomer 2 were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT(2C)R using an intracellular calcium (Ca(i) (2+)) release assay. Compound 1 demonstrated efficacy and potency as an allosteric modulator for the 5-HT(2C)R with no intrinsic agonist activity. Compound 1 did not alter 5-HT-evoked Ca(i) (2+) in CHO cells stably transfected with the highly homologous 5-HT(2A)R. In contrast, the diastereomer 2 did not alter 5-HT-evoked Ca(i) (2+) release in 5-HT(2A)R-CHO or 5-HT(2C)R-CHO cells or exhibit intrinsic agonist activity.

  12. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: hit-to-lead optimization.

    PubMed

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kiss, Béla; Kolok, Sándor; Fónagy, Katalin; Gyertyán, István; Háda, Viktor; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2014-08-15

    An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled the lead criteria.

  13. Computer-aided design of negative allosteric modulators of metabotropic glutamate receptor 5 (mGluR5): Comparative molecular field analysis of aryl ether derivatives.

    PubMed

    Selvam, Chelliah; Thilagavathi, Ramasamy; Narasimhan, Balasubramanian; Kumar, Pradeep; Jordan, Brian C; Ranganna, Kasturi

    2016-02-15

    The metabotropic glutamate receptors (mGlu receptors) have emerged as attractive targets for number of neurological and psychiatric disorders. Recently, mGluR5 negative allosteric modulators (NAMs) have gained considerable attention in pharmacological research. Comparative molecular field analysis (CoMFA) was performed on 73 analogs of aryl ether which were reported as mGluR5 NAMs. The study produced a statistically significant model with high correlation coefficient and good predictive abilities.

  14. Flexible modulation of agonist efficacy at the human A3 adenosine receptor by the imidazoquinoline allosteric enhancer LUF6000

    PubMed Central

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

    2008-01-01

    Background A series of 1H-imidazo- [4,5-c]quinolin-4-amine derivatives, represented by LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo [4,5-c]quinolin-4-amine), are allosteric modulators of the human A3 adenosine receptor (AR). Here we studied the modulation by LUF6000 of the maximum effect (Emax) of structurally diverse agonists at the A3 AR stably expressed in CHO cells. Results In an assay of [35S]GTPγS binding, the Emax of the A3 AR agonist Cl-IB-MECA at the A3 AR was lower than that of the non-selective AR agonist NECA. LUF6000 exerted an Emax-enhancing effect at a concentration of 0.1 μM or higher, and was shown to increase the Emax of Cl-IB-MECA and other low-efficacy agonists to a larger extent than that of the high-efficacy agonist NECA. Interestingly, LUF6000 converted a nucleoside A3 AR antagonist MRS542, but not a non-nucleoside antagonist MRS1220, into an agonist. LUF6000 alone did not show any effect. Mathematical modeling was performed to explain the differential effects of LUF6000 on agonists with various Emax. A simple explanation for the observation that LUF6000 has a much stronger effect on Cl-IB-MECA than on NECA derived from the mathematical modeling is that NECA has relatively strong intrinsic efficacy, such that the response is already close to the maximum response. Therefore, LUF6000 cannot enhance Emax much further. Conclusion LUF6000 was found to be an allosteric enhancer of Emax of structurally diverse agonists at the A3 AR, being more effective for low-Emax agonists than for high-Emax agonists. LUF6000 was demonstrated to convert an antagonist into an agonist, which represents the first example in G protein-coupled receptors. The observations from the present study are consistent with that predicted by mathematical modeling. PMID:19077268

  15. Preclinical pharmacokinetic and toxicological evaluation of MIF-1 peptidomimetic, PAOPA: examining the pharmacology of a selective dopamine D2 receptor allosteric modulator for the treatment of schizophrenia.

    PubMed

    Tan, Mattea L; Basu, Dipannita; Kwiecien, Jacek M; Johnson, Rodney L; Mishra, Ram K

    2013-04-01

    Schizophrenia is a mental illness characterized by a breakdown in cognition and emotion. Over the years, drug treatment for this disorder has mainly been compromised of orthosteric ligands that antagonize the active site of the dopamine D2 receptor. However, these drugs are limited in their use and often lead to the development of adverse movement and metabolic side effects. Allosteric modulators are an emerging class of therapeutics with significant advantages over orthosteric ligands, including an improved therapeutic and safety profile. This study investigates our newly developed allosteric modulator, PAOPA, which is a specific modulator of the dopamine D2 receptor. Previous studies have shown PAOPA to attenuate schizophrenia-like behavioral abnormalities in preclinical models. To advance this newly developed allosteric drug from the preclinical to clinical stage, this study examines the pharmacokinetic behavior and toxicological profile of PAOPA. Results from this study prove the effectiveness of PAOPA in reaching the implicated regions of the brain for therapeutic action, particularly the striatum. Pharmacokinetic parameters of PAOPA were found to be comparable to current market antipsychotic drugs. Necropsy and histopathological analyses showed no abnormalities in all examined organs. Acute and chronic treatment of PAOPA indicated no movement abnormalities commonly found with the use of current typical antipsychotic drugs. Moreover, acute and chronic PAOPA treatment revealed no hematological or metabolic abnormalities classically found with the use of atypical antipsychotic drugs. Findings from this study demonstrate a better safety profile of PAOPA, and necessitates the progression of this newly developed therapeutic for the treatment of schizophrenia.

  16. Identification and characterization of mGlu3 ligands using a high throughput FLIPR assay for detection of agonists, antagonists, and allosteric modulators.

    PubMed

    Pratt, Steven D; Mezler, Mario; Geneste, Hervé; Bakker, Margot H M; Hajduk, Philip J; Gopalakrishnan, Sujatha M

    2011-08-01

    When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (G(α16)), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC(10) and then EC(80) concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands. PMID:21534916

  17. 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

  18. 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

  19. 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.

  20. Structural Mechanisms of Peptide Recognition and Allosteric Modulation of Gene Regulation by the RRNPP Family of Quorum-Sensing Regulators.

    PubMed

    Do, Hackwon; Kumaraswami, Muthiah

    2016-07-17

    The members of RRNPP family of bacterial regulators sense population density-specific secreted oligopeptides and modulate the expression of genes involved in cellular processes, such as sporulation, competence, virulence, biofilm formation, conjugative plasmid transfer and antibiotic resistance. Signaling by RRNPP regulators include several steps: generation and secretion of the signaling oligopeptides, re-internalization of the signaling molecules into the cytoplasm, signal sensing by the cytosolic RRNPP regulators, signal-specific allosteric structural changes in the regulators, and interaction of the regulators with their respective regulatory target and gene regulation. The recently determined structures of the RRNPP regulators provide insight into the mechanistic aspects for several steps in this signaling circuit. In this review, we discuss the structural principles underlying peptide specificity, regulatory target recognition, and ligand-induced allostery in RRNPP regulators and its impact on gene regulation. Despite the conserved tertiary structure of these regulators, structural analyses revealed unexpected diversity in the mechanism of activation and molecular strategies that couple the peptide-induced allostery to gene regulation. Although these structural studies provide a sophisticated understanding of gene regulation by RRNPP regulators, much needs to be learned regarding the target DNA binding by yet-to-be characterized RNPP regulators and the several aspects of signaling by Rgg regulators. PMID:27283781

  1. 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.

  2. Mapping the Binding of GluN2B-Selective N-Methyl-d-aspartate Receptor Negative Allosteric Modulators

    PubMed Central

    Yuan, Hongjie; Karakas, Erkan; Geballe, Matthew; Furukawa, Hiro; Liotta, Dennis C.; Snyder, James P.; Traynelis, Stephen F.

    2012-01-01

    We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists. PMID:22596351

  3. 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.

  4. 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

  5. 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

  6. Galantamine, an acetylcholinesterase inhibitor and positive allosteric modulator of nicotinic acetylcholine receptors, attenuates nicotine taking and seeking in rats.

    PubMed

    Hopkins, Thomas J; Rupprecht, Laura E; Hayes, Matthew R; Blendy, Julie A; Schmidt, Heath D

    2012-09-01

    Current smoking cessation pharmacotherapies have limited efficacy in preventing relapse and maintaining abstinence during withdrawal. Galantamine is an acetylcholinesterase inhibitor that also acts as a positive allosteric modulator of nicotinic acetylcholine receptors. Galantamine has recently been shown to reverse nicotine withdrawal-induced cognitive impairments in mice, which suggests that galantamine may function to prevent relapse in human smokers. However, there are no studies examining whether galantamine administration modulates nicotine self-administration and/or reinstatement of nicotine seeking in rodents. The present experiments were designed to determine the effects of galantamine administration on nicotine taking and reinstatement of nicotine-seeking behavior, an animal model of relapse. Moreover, the effects of galantamine on sucrose-maintained responding and sucrose seeking were also examined to determine whether galantamine's effects generalized to other reinforced behaviors. An inverted U-shaped dose-response curve was obtained when animals self-administered different unit doses of nicotine with the highest responding for 0.03 mg/kg per infusion of nicotine. Acute galantamine administration (5.0 mg/kg, i.p.) attenuated nicotine self-administration when animals were maintained on either a fixed-ratio 5 (FR5) or progressive ratio (PR) schedule of reinforcement. Galantamine administration also attenuated the reinstatement of nicotine-seeking behavior. No significant effects of galantamine on sucrose self-administration or sucrose reinstatement were noted. Acetylcholinesterase inhibitors have also been shown to produce nausea and vomiting in humans. However, at doses required to attenuate nicotine self-administration, no effects of galantamine on nausea/malaise as measured by pica were noted. These results indicate that increased extracellular acetylcholine levels and/or nicotinic acetylcholine receptor stimulation is sufficient to attenuate

  7. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization.

    PubMed

    Hurst, Raymond S; Hajós, Mihaly; Raggenbass, Mario; Wall, Theron M; Higdon, Nicole R; Lawson, Judy A; Rutherford-Root, Karen L; Berkenpas, Mitchell B; Hoffmann, W E; Piotrowski, David W; Groppi, Vincent E; Allaman, Geraldine; Ogier, Roch; Bertrand, Sonia; Bertrand, Daniel; Arneric, Stephen P

    2005-04-27

    Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the alpha7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human alpha7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by alpha4beta2, alpha3beta4, and alpha9alpha10 nAChRs. PNU-120596 increased the channel mean open time of alpha7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of alpha7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances alpha7 nAChR function and thus has the potential to treat psychiatric and neurological disorders. PMID:15858066

  8. 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

  9. 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…

  10. 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.

  11. Interactions of allosteric modulators of AMPA/kainate receptors on spreading depression in the chicken retina.

    PubMed

    Kertész, Szabolcs; Kapus, Gábor; Lévay, György

    2004-10-29

    The functional role of AMPA and kainate receptors in spreading depression (SD) was investigated in the isolated chicken retina. Competitive (NBQX) and non-competitive (GYKI 52466, GYKI 53405 and GYKI 53655) antagonists of the AMPA receptor inhibited AMPA-induced SD in a concentration-dependent manner. Concentrations of drugs caused 50% inhibition (IC(50) values) are 0.2, 16.6, 7.0 and 1.4 microM, respectively. AMPA receptor positive modulator cyclothiazide was more effective in the potentiation of SD evoked by AMPA than by kainate. Slight potentiation of either AMPA- or kainate-induced SD was observed only at high concentration (1 mg/ml) by the kainate receptor modulator concanavalin A. Compounds that positively modulate AMPA receptor function (cyclothiazide, IDRA-21, S 18986, 1-BCP and aniracetam) caused a concentration-dependent potentiation in SD. Concentrations of drugs that caused 50% potentiation (estimated EC(50) values) are 9, 135, 142, 450 and 1383 microM, respectively. Interaction between cyclothiazide, aniracetam or S 18986 administered with each other, or with GYKI 52466, respectively, was also investigated. When cyclothiazide and S 18986 were co-applied, their effects seemed to be additive. However, lack of additivity was obtained when S 18986 was added together with aniracetam. Positive modulators applied at equiactive concentrations reduced the inhibitory action of GYKI 52466 and differently shifted its concentration-response curve. In this respect, S 18986 was the most effective (IC(50) of GYKI 52466 changed from 16.6 to 51.9 microM). Our findings indicate the contribution of AMPA rather than kainate receptors in the mediation of retinal spreading depression. Our data further support the idea that multiple positive modulatory sites are present on the AMPA receptor complex in addition to a negative modulatory site.

  12. 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

  13. 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. PMID:27661904

  14. 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. PMID:27474687

  15. Development of a novel, CNS-penetrant, metabotropic glutamate receptor 3 (mGlu3) NAM probe (ML289) derived from a closely related mGlu5 PAM.

    PubMed

    Sheffler, Douglas J; Wenthur, Cody J; Bruner, Joshua A; Carrington, Sheridan J S; Vinson, Paige N; Gogi, Kiran K; Blobaum, Anna L; Morrison, Ryan D; Vamos, Mitchell; Cosford, Nicholas D P; Stauffer, Shaun R; Daniels, J Scott; Niswender, Colleen M; Conn, P Jeffrey; Lindsley, Craig W

    2012-06-15

    Herein we report the discovery and SAR of a novel metabotropic glutamate receptor 3 (mGlu(3)) NAM probe (ML289) with 15-fold selectivity versus mGlu(2). The mGlu(3) NAM was discovered via a 'molecular switch' from a closely related, potent mGlu(5) positive allosteric modulator (PAM), VU0092273. This NAM (VU0463597, ML289) displays an IC(50) value of 0.66 μM and is inactive against mGlu(5). PMID:22607673

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

    PubMed Central

    Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J.; Panarese, Joseph D.; Niswender, Colleen; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    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. PMID:25435150

  17. Amyloid-β peptides act as allosteric modulators of cholinergic signalling through formation of soluble BAβACs.

    PubMed

    Kumar, Rajnish; Nordberg, Agneta; Darreh-Shori, Taher

    2016-01-01

    -β interacts readily in an apolipoprotein-facilitated manner with butyrylcholinesterase, forming highly stable and soluble complexes, BAβACs, which can be separated in their native states by sucrose density gradient technique. Enzymological analyses further evinced that amyloid-β concentration dependently increased the acetylcholine-hydrolyzing capacity of cholinesterases. In silico biomolecular analysis further deciphered the allosteric amino acid fingerprint of the amyloid-β-cholinesterase molecular interaction in formation of BAβACs. In the case of butyrylcholinesterase, the results indicated that amyloid-β interacts with a putative activation site at the mouth of its catalytic tunnel, most likely leading to increased acetylcholine influx into the catalytic site, and thereby increasing the intrinsic catalytic rate of butyrylcholinesterase. In conclusion, at least one of the native physiological functions of amyloid-β is allosteric modulation of the intrinsic catalytic efficiency of cholinesterases, and thereby regulation of synaptic and extrasynaptic cholinergic signalling. High apolipoprotein-E may pathologically alter the biodynamics of this amyloid-β function.

  18. Amyloid-β peptides act as allosteric modulators of cholinergic signalling through formation of soluble BAβACs

    PubMed Central

    Kumar, Rajnish; Nordberg, Agneta

    2016-01-01

    amyloid-β interacts readily in an apolipoprotein-facilitated manner with butyrylcholinesterase, forming highly stable and soluble complexes, BAβACs, which can be separated in their native states by sucrose density gradient technique. Enzymological analyses further evinced that amyloid-β concentration dependently increased the acetylcholine-hydrolyzing capacity of cholinesterases. In silico biomolecular analysis further deciphered the allosteric amino acid fingerprint of the amyloid-β-cholinesterase molecular interaction in formation of BAβACs. In the case of butyrylcholinesterase, the results indicated that amyloid-β interacts with a putative activation site at the mouth of its catalytic tunnel, most likely leading to increased acetylcholine influx into the catalytic site, and thereby increasing the intrinsic catalytic rate of butyrylcholinesterase. In conclusion, at least one of the native physiological functions of amyloid-β is allosteric modulation of the intrinsic catalytic efficiency of cholinesterases, and thereby regulation of synaptic and extrasynaptic cholinergic signalling. High apolipoprotein-E may pathologically alter the biodynamics of this amyloid-β function. PMID:26525916

  19. Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain.

    PubMed

    Petrel, Christophe; Kessler, Albane; Dauban, Philippe; Dodd, Robert H; Rognan, Didier; Ruat, Martial

    2004-04-30

    A three-dimensional model of the human extracellular Ca(2+)-sensing receptor (CaSR) has been used to identify specific residues implicated in the recognition of two negative allosteric CaSR modulators of different chemical structure, NPS 2143 and Calhex 231. To demonstrate the involvement of these residues, we have analyzed dose-inhibition response curves for the effect of these calcilytics on Ca(2+)-induced [(3)H]inositol phosphate accumulation for the selected CaSR mutants transiently expressed in HEK293 cells. These mutants were further used for investigating the binding pocket of two chemically unrelated positive allosteric CaSR modulators, NPS R-568 and (R)-2-[1-(1-naphthyl)ethylaminomethyl]-1H-indole (Calindol), a novel potent calcimimetic that stimulates (EC(50) = 0.31 microM) increases in [(3)H]inositol phosphate levels elicited by activating the wild-type CaSR by 2 mM Ca(2+). Our data validate the involvement of Trp-818(6.48), Phe-821(6.51), Glu-837(7.39), and Ile-841(7.43) located in transmembranes (TM) 6 and TM7, in the binding pocket for both calcimimetics and calcilytics, despite important differences observed between each family of compounds. The TMs involved in the recognition of both calcilytics include residues located in TM3 (Arg-680(3.28), Phe-684(3.32), and Phe-688(3.36)). However, our study indicates subtle differences between the binding of these two compounds. Importantly, the observation that some mutations that have no effect on calcimimetics recognition but which affect the binding of calcilytics in TM3 and TM5, suggests that the binding pocket of positive and negative allosteric modulators is partially overlapping but not identical. Our CaSR model should facilitate the development of novel drugs of this important therapeutic target and the identification of the molecular determinants involved in the binding of allosteric modulators of class 3 G-protein-coupled receptors. PMID:14976203

  20. Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake.

    PubMed

    Zhang, Peng; Li, Shufen; Gao, Yang; Lu, Wenbo; Huang, Ke; Ye, Deyong; Li, Xi; Chu, Yong

    2014-12-15

    Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC50=8 μM) and BTO-5s (IC50=10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.

  1. Maximizing the effect of an α7 nicotinic receptor PAM in a mouse model of schizophrenia-like sensory inhibition deficits.

    PubMed

    Stevens, Karen E; Zheng, Lijun; Floyd, Kirsten L; Stitzel, Jerry A

    2015-06-22

    Positive allosteric modulators (PAMs) for the α7 nicotinic receptor hold promise for the treatment of sensory inhibition deficits observed in schizophrenia patients. Studies of these compounds in the DBA/2 mouse, which models the schizophrenia-related deficit in sensory inhibition, have shown PAMs to be effective in improving the deficit. However, the first published clinical trial of a PAM for both sensory inhibition deficits and related cognitive difficulties failed, casting a shadow on this therapeutic approach. The present study used both DBA/2 mice, and C3H Chrna7 heterozygote mice to assess the ability of the α7 PAM, PNU-120596, to improve sensory inhibition. Both of these strains of mice have reduced hippocampal α7 nicotinic receptor numbers and deficient sensory inhibition similar to schizophrenia patients. Low doses of PNU-120596 (1 or 3.33mg/kg) were effective in the DBA/2 mouse but not the C3H Chrna7 heterozygote mouse. Moderate doses of the selective α7 nicotinic receptor agonist, choline chloride (10 or 33mg/kg), were also ineffective in improving sensory inhibition in the C3H Chrna7 heterozygote mouse. However, combining the lowest doses of both PNU-120596 and choline chloride in this mouse model did improve sensory inhibition. We propose here that the difference in efficacy of PNU-120596 between the 2 mouse strains is driven by differences in hippocampal α7 nicotinic receptor numbers, such that C3H Chrna7 heterozygote mice require additional direct stimulation of the α7 receptors. These data may have implications for further clinical testing of putative α7 nicotinic receptor PAMs.

  2. 5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

    PubMed

    Massink, Arnault; Louvel, Julien; Adlere, Ilze; van Veen, Corine; Huisman, Berend J H; Dijksteel, Gabrielle S; Guo, Dong; Lenselink, Eelke B; Buckley, Benjamin J; Matthews, Hayden; Ranson, Marie; Kelso, Michael; IJzerman, Adriaan P

    2016-05-26

    The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site. PMID:27124340

  3. Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences.

    PubMed

    Choudhury, Susobhan; Naiya, Gitashri; Singh, Priya; Lemmens, Peter; Roy, Siddhartha; Pal, Samir Kumar

    2016-04-01

    Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond-resolved FRET and polarisation-gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences OE and OI . FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C-terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation-gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein-protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with OE and OI with the consequent protein-protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment.

  4. Positive or negative allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) does not alter expression of behavioral sensitization to methamphetamine.

    PubMed

    Kufahl, Peter R; Nemirovsky, Natali E; Watterson, Lucas R; Zautra, Nicholas; Olive, M Foster

    2013-01-01

    We investigated the role of metabotropic glutamate receptor type 5 (mGluR5) in methamphetamine-induced behavioral sensitization. The mGluR5 positive allosteric modulator (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) and negative allosteric modulator fenobam were tested in separate experiments. Sprague-Dawley rats were repeatedly injected with 1 mg/kg methamphetamine or saline, and then given a locomotor challenge test using a dose of 0.5 mg/kg methamphetamine. Prior to the challenge test session, rats were injected with CDPPB, fenobam, or a vehicle.  Doses from previous studies showed reduced drug-conditioned behavior; however in this study neither CDPPB nor fenobam pretreatment resulted in an altered expression of behavioral sensitization, indicating a lack of mGluR5 involvement in sensitized methamphetamine-induced locomotion. Additionally, the high dose (30 mg/kg) of fenobam resulted in decreased methamphetamine-induced locomotion in rats regardless of drug exposure history, which suggests evidence of nonspecific behavioral inhibition.

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

    PubMed

    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

  6. 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

  7. 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

    German, Nadezhda; Decker, Ann M; Gilmour, Brian P; Gay, Elaine A; Wiley, Jenny L; Thomas, Brian F; Zhang, Yanan

    2014-09-25

    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 [(3)H]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.

  8. The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA

    PubMed Central

    Ahmed, Ahmed H.; Ptak, Christopher P.; Oswald, Robert E.

    2011-01-01

    Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes. PMID:20199107

  9. Synthesis and biological evaluation of a new series of 2-amino-3-aroyl thiophene derivatives as agonist allosteric modulators of the A1 adenosine receptor. A position-dependent effect study.

    PubMed

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Lopez-Cara, Carlota; Cruz-Lopez, Olga; Moorman, Allan R; Massink, Arnault; IJzerman, Adriaan P; Vincenzi, Fabrizio; Borea, Pier Andrea; Varani, Katia

    2015-08-28

    The 2-amino-3-(p-chlorobenzoyl)thiophene scaffold has been widely employed as a pharmacophore for the identification of small molecules acting as allosteric modulators at the adenosine A1 receptor. A new series of 2-amino-3-(p-chlorobenzoyl)-4-benzyl-5-arylthiophene derivatives, characterized by the absence as well as the presence of electron-releasing or electron-withdrawing groups on the phenyl ring at the 4- and 5-positions of the thiophene ring, were identified as positive allosteric enhancers at the adenosine A1 receptor in binding (saturation, competition and dissociation kinetics) and functional assays. To better understand the positional requirements of substituents on the 2-amino-3-(p-chlorobenzoyl)thiophene core, the corresponding regioisomeric 4-aryl-5-benzylthiophene analogues were synthesized and found to possess reduced allosteric enhancer activity. PMID:26141910

  10. Unraveling structural mechanisms of allosteric drug action.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung

    2014-05-01

    Orthosteric drugs block the active site to obstruct function; allosteric drugs modify the population of the active state, to modulate function. Available data lead us to propose that allosteric drugs can constitute anchors and drivers. The anchor docks into an allosteric pocket. The conformation with which it interacts is unchanged during the transition between the inactive and active states. The anchor provides the foundation that allows the driver to exert a 'pull' and/or 'push' action that shifts the receptor population from the inactive to the active state. The presence or absence of driver atom in an allosteric drug can exert opposite agonism. We map a strategy for driver identification and expect the allosteric trigger concept to transform agonist/antagonist drug discovery.

  11. Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders.

    PubMed

    Babinsky, Valerie N; Hannan, Fadil M; Gorvin, Caroline M; Howles, Sarah A; Nesbit, M Andrew; Rust, Nigel; Hanyaloglu, Aylin C; Hu, Jianxin; Spiegel, Allen M; Thakker, Rajesh V

    2016-05-13

    Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+) i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca(2+) i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca(2+) i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+) o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+) i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca(2+) o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling

  12. 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

  13. Recent computational advances in the identification of allosteric sites in proteins.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Zhang, Jian

    2014-10-01

    Allosteric modulators have the potential to fine-tune protein functional activity. Therefore, the targeting of allosteric sites, as a strategy in drug design, is gaining increasing attention. Currently, it is not trivial to find and characterize new allosteric sites by experimental approaches. Alternatively, computational approaches are useful in helping researchers analyze and select potential allosteric sites for drug discovery. Here, we review state-of-the-art computational approaches directed at predicting putative allosteric sites in proteins, along with examples of successes in identifying allosteric sites utilizing these methods. We also discuss the challenges in developing reliable methods for predicting allosteric sites and tactics to resolve demanding tasks. PMID:25107670

  14. Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

    PubMed

    Kalinichev, Mikhail; Le Poul, Emmanuel; Boléa, Christelle; Girard, Françoise; Campo, Brice; Fonsi, Massimiliano; Royer-Urios, Isabelle; Browne, Susan E; Uslaner, Jason M; Davis, Matthew J; Raber, Jacob; Duvoisin, Robert; Bate, Simon T; Reynolds, Ian J; Poli, Sonia; Celanire, Sylvain

    2014-09-01

    There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis. PMID:24947466

  15. Characterization of the Novel Positive Allosteric Modulator of the Metabotropic Glutamate Receptor 4 ADX88178 in Rodent Models of Neuropsychiatric Disorders

    PubMed Central

    Le Poul, Emmanuel; Boléa, Christelle; Girard, Françoise; Campo, Brice; Fonsi, Massimiliano; Royer-Urios, Isabelle; Browne, Susan E.; Uslaner, Jason M.; Davis, Matthew J.; Raber, Jacob; Duvoisin, Robert; Bate, Simon T.; Reynolds, Ian J.; Celanire, Sylvain

    2014-01-01

    There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]–induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis. PMID:24947466

  16. Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

    PubMed

    Kalinichev, Mikhail; Le Poul, Emmanuel; Boléa, Christelle; Girard, Françoise; Campo, Brice; Fonsi, Massimiliano; Royer-Urios, Isabelle; Browne, Susan E; Uslaner, Jason M; Davis, Matthew J; Raber, Jacob; Duvoisin, Robert; Bate, Simon T; Reynolds, Ian J; Poli, Sonia; Celanire, Sylvain

    2014-09-01

    There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.

  17. Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation.

    PubMed

    Rüsch, Dirk; Zhong, Huijun; Forman, Stuart A

    2004-05-14

    At clinical concentrations, the potent intravenous general anesthetic etomidate enhances gamma-aminobutyric acid, type A (GABA(A)) receptor activity elicited with low gamma-aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA(A) receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channel-activating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(+)-etomidate isomer and display parallel dependence on GABA(A) beta subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human alpha1beta2gamma2L receptors. Etomidate at up to 100 microm reduced GABA EC(50) values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC(50) and increased maximal efficacy, demonstrating that etomidate shifts the GABA(A) receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux co-agonist mechanism with two equivalent etomidate sites that allosterically enhance GABA(A) receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA(A) receptors containing a point mutation that increases constitutive gating activity.

  18. Chemical Modulation of Mutant mGlu1 Receptors Derived from Deleterious GRM1 Mutations Found in Schizophrenics

    PubMed Central

    2014-01-01

    Schizophrenia is a complex and highly heterogeneous psychiatric disorder whose precise etiology remains elusive. While genome-wide association studies (GWAS) have identified risk genes, they have failed to determine if rare coding single nucleotide polymorphisms (nsSNPs) contribute in schizophrenia. Recently, two independent studies identified 12 rare, deleterious nsSNPS in the GRM1 gene, which encodes the metabotropic glutamate receptor subtype 1 (mGlu1), in schizophrenic patients. Here, we generated stable cell lines expressing the mGlu1 mutant receptors and assessed their pharmacology. Using both the endogenous agonist glutamate and the synthetic agonist DHPG, we found that several of the mutant mGlu1 receptors displayed a loss of function that was not due to a loss in plasma membrane expression. Due to a lack of mGlu1 positive allosteric modulators (PAM) tool compounds active at human mGlu1, we optimized a known mGlu4 PAM/mGlu1 NAM chemotype into a series of potent and selective mGlu1 PAMs by virtue of a double “molecular switch”. Employing mGlu1 PAMs from multiple chemotypes, we demonstrate that the mutant receptors can be potentiated by small molecules and in some cases efficacy restored to that comparable to wild type mGlu1 receptors, suggesting deficits in patients with schizophrenia due to these mutations may be amenable to intervention with an mGlu1 PAM. However, in wild type animals, mGlu1 negative allosteric modulators (NAMs) are efficacious in classic models predictive of antipsychotic activity, whereas we show that mGlu1 PAMs have no effect to slight potentiation in these models. These data further highlight the heterogeneity of schizophrenia and the critical role of patient selection strategies in psychiatric clinical trials to match genotype with therapeutic mechanism. PMID:25137254

  19. Escherichia coli SufE sulfur transfer protein modulates the SufS cysteine desulfurase through allosteric conformational dynamics.

    PubMed

    Singh, Harsimran; Dai, Yuyuan; Outten, F Wayne; Busenlehner, Laura S

    2013-12-20

    Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from L-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.

  20. Age-related testicular toxicity of mGluR5 negative allosteric modulators appears to be unrelated to testis drug transporter maturity.

    PubMed

    Campion, Sarah N; Marcek, John M; Kumpf, Steven W; Chapin, Robert E; Houle, Christopher; Cappon, Gregg D

    2015-04-01

    Testicular degeneration was observed in exploratory toxicity studies in Wistar rats treated with several mGluR5 negative allosteric modulators. To determine if these testis effects were influenced by animal age, these compounds were administered to male Wistar rats of different ages (8, 10, and 12 weeks old) for 2 weeks followed by evaluation of male reproductive organ weights, testis histopathology, and inhibin B levels. Overall, seminiferous tubule degeneration was observed in 2/15, 5/15, and 0/15 compound treated rats from the 8, 10, and 12 week old cohorts and inhibin B was decreased in 8 and 10 week old animals, but not in 12 week old rats, suggesting that there is an age-related component to this testis toxicity. The gene expression profiles of drug transporters in the testis of rats aged PND 38 through PND 91 were very similar, indicating that immaturity of these transporters is an unlikely factor contributing to the age-related toxicity.

  1. Effects of a metabotropic glutamate receptor subtype 7 negative allosteric modulator in the periaqueductal grey on pain responses and rostral ventromedial medulla cell activity in rat.

    PubMed

    Palazzo, Enza; Marabese, Ida; Luongo, Livio; Boccella, Serena; Bellini, Giulia; Giordano, Maria Elvira; Rossi, Francesca; Scafuro, Mariantonietta; Novellis, Vito de; Maione, Sabatino

    2013-09-03

    The metabotropic glutamate receptor 7 (mGluR7) negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), was locally microinjected into the ventrolateral periaqueductal gray (VL PAG) and the effect on pain responses in formalin and spare nerve injury (SNI) -induced neuropathic pain models was monitored in the rat. The activity of rostral ventromedial medulla (RVM) "pronociceptive" ON and "antinociceptive" OFF cells was also evaluated. Intra-VL PAG MMPIP blocked the first and second phase of nocifensive behaviour in the formalin pain model. MMPIP increased the tail flick latency and simultaneously increased the activity of the OFF cells while inhibiting that of ON cells in rats with SNI of the sciatic nerve. MMPIP failed to modify nociceptive responses and associated RVM ON and OFF cell activity in sham rats. An increase in mGluR7 gene, protein and staining, the latter being associated with vesicular glutamate transporter-positive profiles, has been found in the VL PAG in SNI rats. Blockade of mGluR7 within the VL PAG has an antinociceptive effect in formalin and neuropathic pain models. VL PAG mGluR7 blockade offers a target for dis-inhibiting the VL PAG-RVM pathway and silencing pain in inflammatory and neuropathic pain models.

  2. 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.

  3. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

    PubMed

    Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

    2016-10-01

    Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.

  4. 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

  5. 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

  6. Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor.

    PubMed

    Petrel, Christophe; Kessler, Albane; Maslah, Fouzia; Dauban, Philippe; Dodd, Robert H; Rognan, Didier; Ruat, Martial

    2003-12-01

    A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors. PMID:14506236

  7. Pharmacology of basimglurant (RO4917523, RG7090), a unique metabotropic glutamate receptor 5 negative allosteric modulator in clinical development for depression.

    PubMed

    Lindemann, Lothar; Porter, Richard H; Scharf, Sebastian H; Kuennecke, Basil; Bruns, Andreas; von Kienlin, Markus; Harrison, Anthony C; Paehler, Axel; Funk, Christoph; Gloge, Andreas; Schneider, Manfred; Parrott, Neil J; Polonchuk, Liudmila; Niederhauser, Urs; Morairty, Stephen R; Kilduff, Thomas S; Vieira, Eric; Kolczewski, Sabine; Wichmann, Juergen; Hartung, Thomas; Honer, Michael; Borroni, Edilio; Moreau, Jean-Luc; Prinssen, Eric; Spooren, Will; Wettstein, Joseph G; Jaeschke, Georg

    2015-04-01

    Major depressive disorder (MDD) is a serious public health burden and a leading cause of disability. Its pharmacotherapy is currently limited to modulators of monoamine neurotransmitters and second-generation antipsychotics. Recently, glutamatergic approaches for the treatment of MDD have increasingly received attention, and preclinical research suggests that metabotropic glutamate receptor 5 (mGlu5) inhibitors have antidepressant-like properties. Basimglurant (2-chloro-4-[1-(4-fluoro-phenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]-pyridine) is a novel mGlu5 negative allosteric modulator currently in phase 2 clinical development for MDD and fragile X syndrome. Here, the comprehensive preclinical pharmacological profile of basimglurant is presented with a focus on its therapeutic potential for MDD and drug-like properties. Basimglurant is a potent, selective, and safe mGlu5 inhibitor with good oral bioavailability and long half-life supportive of once-daily administration, good brain penetration, and high in vivo potency. It has antidepressant properties that are corroborated by its functional magnetic imaging profile as well as anxiolytic-like and antinociceptive features. In electroencephalography recordings, basimglurant shows wake-promoting effects followed by increased delta power during subsequent non-rapid eye movement sleep. In microdialysis studies, basimglurant had no effect on monoamine transmitter levels in the frontal cortex or nucleus accumbens except for a moderate increase of accumbal dopamine, which is in line with its lack of pharmacological activity on monoamine reuptake transporters. These data taken together, basimglurant has favorable drug-like properties, a differentiated molecular mechanism of action, and antidepressant-like features that suggest the possibility of also addressing important comorbidities of MDD including anxiety and pain as well as daytime sleepiness and apathy or lethargy.

  8. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  9. An acetylcholine alpha7 positive allosteric modulator rescues a schizophrenia-associated brain endophenotype in the 15q13.3 microdeletion, encompassing CHRNA7.

    PubMed

    Gass, Natalia; Weber-Fahr, Wolfgang; Sartorius, Alexander; Becker, Robert; Didriksen, Michael; Stensbøl, Tine Bryan; Bastlund, Jesper Frank; Meyer-Lindenberg, Andreas; Schwarz, Adam J

    2016-07-01

    The 15q13.3 microdeletion copy number variation is strongly associated with schizophrenia and epilepsy. The CHRNA7 gene, encoding nicotinic acetylcholine alpha 7 receptors (nAChA7Rs), is hypothesized to be one of the main genes in this deletion causing the neuropsychiatric phenotype. Here we used a recently developed 15q13.3 microdeletion mouse model to explore whether an established schizophrenia-associated connectivity phenotype is replicated in a murine model, and whether positive modulation of nAChA7 receptor might pharmacologically normalize the connectivity patterns. Resting-state fMRI data were acquired from male mice carrying a hemizygous 15q13.3 microdeletion (N=9) and from wild-type mice (N=9). To study the connectivity profile of 15q13.3 mice and test the effect of nAChA7 positive allosteric modulation, the 15q13.3 mice underwent two imaging sessions, one week apart, receiving a single intraperitoneal injection of either 15mg/kg Lu AF58801 or saline. The control group comprised wild-type mice treated with saline. We performed seed-based functional connectivity analysis to delineate aberrant connectivity patterns associated with the deletion (15q13.3 mice (saline treatment) versus wild-type mice (saline treatment)) and their modulation by Lu AF58801 (15q13.3 mice (Lu AF58801 treatment) versus 15q13.3 mice (saline treatment)). Compared to wild-type mice, 15q13.3 mice evidenced a predominant hyperconnectivity pattern. The main effect of Lu AF58801 was a normalization of elevated functional connectivity between prefrontal and frontal, hippocampal, striatal, thalamic and auditory regions. The strongest effects were observed in brain regions expressing nAChA7Rs, namely hippocampus, cerebral cortex and thalamus. These effects may underlie the antiepileptic, pro-cognitive and auditory gating deficit-reversal effects of nAChA7R stimulation. PMID:27061851

  10. 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.

  11. 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

  12. MMPIP, an mGluR7-selective negative allosteric modulator, alleviates pain and normalizes affective and cognitive behavior in neuropathic mice.

    PubMed

    Palazzo, Enza; Romano, Rosaria; Luongo, Livio; Boccella, Serena; De Gregorio, Danilo; Giordano, Maria Elvira; Rossi, Francesca; Marabese, Ida; Scafuro, Maria Antonietta; de Novellis, Vito; Maione, Sabatino

    2015-06-01

    This study investigated the effects of a single administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridinyl-4-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), a negative allosteric modulator (NAM) of metabotropic glutamate receptor 7 (mGluR7), on pain and on affective and cognitive behavior in neuropathic mice. The activity of pyramidal neurons in the prelimbic cortex (PLC), which respond to stimulation of the basolateral amygdala (BLA) with either excitation or inhibition, was also investigated. The spared nerve injury (SNI) of the sciatic nerve induced, 14 days after surgery, thermal hyperalgesia and mechanical allodynia, reduced open-arm choice in the elevated plus-maze, increased time of immobility in the tail suspension, and increased digging and burying in the marble burying test. Cognitive performance was also significantly compromised in the SNI mice. Spared nerve injury induced phenotypic changes on pyramidal neurons of the PLC; excitatory responses increased, whereas inhibitory responses decreased after BLA stimulation. mGluR7 expression, mainly associated with vesicular glutamate transporter, increased in the hippocampus and decreased in the BLA, PLC, and dorsal raphe in SNI mice. MMPIP increased thermal and mechanical thresholds and open-arm choice. It reduced the immobility in the tail suspension test and the number of marbles buried and of digging events in the marble burying test. MMPIP also improved cognitive performance and restored the balance between excitatory and inhibitory responses of PLC neurons in SNI mice. 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one, XAP044, another selective mGluR7 NAM, reproduced the effects of MMPIP on thermal hyperalgesia, mechanical allodynia, tail suspension, and marble burying test. Altogether, these findings show that mGluR7 NAMs reduce pain responses and affective/cognitive impairments in neuropathic pain conditions.

  13. 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.

  14. Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues.

    PubMed

    Rochera, Lourdes; Fresquet, Vicente; Rubio, Vicente; Cervera, Javier

    2002-03-13

    The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes. PMID:11943174

  15. 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...

  16. Positive allosteric modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action.

    PubMed

    Fitzpatrick, Ciarán M; Larsen, Maria; Madsen, Louise H; Caballero-Puntiverio, Maitane; Pickering, Darryl S; Clausen, Rasmus P; Andreasen, Jesper T

    2016-09-01

    Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR). Thus, chronic antidepressant treatment increases AMPAR-mediated neurotransmission and AMPAR-positive allosteric modulators have shown antidepressant-like efficacy in rodents. Here, the effect of enhanced AMPAR neurotransmission on the antidepressant-like and anxiolytic-like actions of the selective serotonin reuptake inhibitor citalopram (0-10 mg/kg) was investigated in mice using the AMPAR-positive allosteric modulator LY451646 (0-3 mg/kg). Antidepressant-like effects were assessed using the forced-swim test (FST), whereas anxiolytic-like effects were tested using the elevated zero maze (EZM) and the marble burying test. LY451646 (3 mg/kg) increased swim distance in the FST and a subactive dose of LY451646 (1 mg/kg) enhanced the effect of citalopram in the FST. In the EZM, LY451646 (3 mg/kg) did not show anxiogenic effects alone, but blocked the anxiolytic-like action of citalopram in the EZM, as reflected by an increase in the latency to enter the open areas and a decrease in the number of entries and time spent in the open areas in citalopram-treated mice. In the marble burying test, LY451646 (3 mg/kg) showed no effect alone, but significantly attenuated the anxiolytic-like effect of citalopram (1.25-2.5 mg/kg) by increasing the number of marbles buried in citalopram-treated mice. These results suggest that AMPAR neurotransmission plays opposite roles in anxiety and depression as AMPAR potentiation facilitated the antidepressant-like effects of citalopram while attenuating its anxiolytic-like effect. These findings have ramifications in the search for AMPAR-based novel anxiolytic and antidepressant treatments. PMID:27341500

  17. Seasonal primary amebic meningoencephalitis (PAM) in the south: summertime is PAM time.

    PubMed

    Diaz, James

    2012-01-01

    Primary amebic meningoencephalitis (PAM), a typically fatal, free-living amebic infection of the central nervous system (CNS), is caused by the thermophilic, freshwater protozoan, Naegleria fowleri. More than 145 cases of PAM have been reported worldwide, with most reported cases in the United States (US). Since annual PAM case clusters in the US and worldwide have demonstrated recent increases over background cases, the objectives of this investigation included (1) an epidemiological and statistical analysis of a 2007 cluster of six PAM cases in the southern US, nested in a retrospective review of 121 confirmed US cases of PAM over the period, 1937 to 2007; and (2) a statistical analysis of all existing demographic, temporal, and behavioral risk factors for PAM. Significant risk factors for PAM in the United States included male sex and warm recreational freshwater exposures in seasonal patterns (July - August) in southern tier states, including Louisiana. Although there have been a few recent survivors of PAM treated with combinations of intensive critical care, antifungals, and synergistic antibiotics, case fatality rates for PAM remain very high. PAM is best prevented by combinations of public health educational and behavioral modification strategies. Further investigations will be required to determine the significance of freshwater wakeboarding as a significant risk factor for PAM and to determine any dose-response effects of global warming on rising freshwater temperatures and the growth of aquatic Naegleria fowleri.

  18. Seven transmembrane receptors as nature's prototype allosteric protein: de-emphasizing the geography of binding.

    PubMed

    Kenakin, Terry P

    2008-09-01

    The article in this issue by Redka et al. (p. 834) illustrates some interesting interactions between classified orthosteric (bind to the same recognition site as endogenous agonist) and allosteric (bind to a different site) ligands. Of particular interest are the methods used to deal with an obfuscating factor in these kinds of studies, namely the propensity of seven transmembrane receptors to form dimers and thus demonstrate allosteric effects through binding at the orthosteric site. The judicious use of kinetics to detect and quantify allosteric action also is demonstrated. The various unique properties of allosteric modulators are discussed in the context of the increasing prevalence of allosteric ligands as investigational drugs.

  19. Efficacy and safety of an adjunctive mGlu2 receptor positive allosteric modulator to a SSRI/SNRI in anxious depression.

    PubMed

    Kent, Justine M; Daly, Ella; Kezic, Iva; Lane, Rosanne; Lim, Pilar; De Smedt, Heidi; De Boer, Peter; Van Nueten, Luc; Drevets, Wayne C; Ceusters, Marc

    2016-06-01

    This phase 2a, randomized, multicenter, double-blind, proof-of-concept study was designed to evaluate, efficacy, safety and tolerability of JNJ-40411813/ADX71149, a novel metabotropic glutamate 2 receptor positive allosteric modulator as an adjunctive treatment for major depressive disorder (MDD) with significant anxiety symptoms. Eligible patients (18-64 years) had a DSM-IV diagnosis of MDD, Hamilton Depression Rating Scale-17 (HDRS17) score of ≥ 18, HDRS17 anxiety/somatization factor score of ≥ 7, and an insufficient response to current treatment with a selective serotonin reuptake inhibitor or serotonin-norepinephrine reuptake inhibitor. The doubly-randomized, 8-week double-blind treatment phase was comprised of two 4-week periods, from which a combined test statistic was generated, with pre-determined weights assigned to each of the 2 treatment periods. Period 1: patients (n=121) were randomly assigned (1:1) to JNJ-40411813 (n=62; 50mg to 150 mg b.i.d, flexibly dosed) or placebo (n=59); Period 2: placebo-treated patients (n=22) who continued to meet entry severity criteria were re-randomized (1:1) to JNJ-40411813 or placebo, while other patients underwent sham re-randomization and continued on their same treatment. Of 121 randomized patients, 100 patients (82.6%) were completers. No efficacy signal was detected on the primary endpoint, the 6-item Hamilton Anxiety Subscale (HAM-A6, p=0.51). Efficacy signals (based on prespecified 1-sided p<0.20) were evident on several secondary outcome measures of both depression (HDRS17 total score, 6-item subscale of HDRS17 assessing core depressive symptoms [HAM-D6], and Inventory of Depressive Symptomatology [IDS-C30]) and anxiety (HDRS17 anxiety/somatization factor, IDS-C30 anxiety subscale). Although well-tolerated, the results do not suggest efficacy for JNJ-40411813 as an adjunctive treatment for patients with MDD with significant anxious symptoms in the dose range studied.

  20. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    PubMed

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  1. PAMS photo image retrieval prototype alternatives analysis

    SciTech Connect

    Conner, M.L.

    1996-04-30

    Photography and Audiovisual Services uses a system called the Photography and Audiovisual Management System (PAMS) to perform order entry and billing services. The PAMS system utilizes Revelation Technologies database management software, AREV. Work is currently in progress to link the PAMS AREV system to a Microsoft SQL Server database engine to provide photograph indexing and query capabilities. The link between AREV and SQLServer will use a technique called ``bonding.`` This photograph imaging subsystem will interface to the PAMS system and handle the image capture and retrieval portions of the project. The intent of this alternatives analysis is to examine the software and hardware alternatives available to meet the requirements for this project, and identify a cost-effective solution.

  2. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  3. 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

  4. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    PubMed

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  5. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

    PubMed Central

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J.; Stockwell, Simon R.; Stokes, Jamie E.; Tan, Yaw S.; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J.; Hyvönen, Marko; Spring, David R.; McKenzie, Grahame J.; Venkitaraman, Ashok R.

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  6. Pharmacological characterization of cGMP regulation by the biarylpropylsulfonamide class of positive, allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

    PubMed

    Ryder, John W; Falcone, Julie F; Manro, Jason R; Svensson, Kjell A; Merchant, Kalpana M

    2006-10-01

    The biarylpropylsulfonamide class of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson's disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was

  7. 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

  8. 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,...

  9. Hydrodynamic Issues in PAMS Mandrel Target Fabrication

    SciTech Connect

    McQuillan, B M; Paguio, R; Subramanian, P; Takagi, M; Zebib, A

    2003-08-27

    Imperfections in PAMS mandrels critically govern the quality of final ICF targets. Imperfections in the mandrels can have a wide range of origins. Here, they present observations of 3 types of imperfections, and data to support the proposal that hydrodynamic factors during the curing of the mandrel are potential causes of these imperfections.

  10. HYDRODYNAMIC ISSUES IN PAMS MANDREL TARGET FABRICATION

    SciTech Connect

    McQUILLAN,B.W; PAGUIO,R; SUBRAMANIAN,P; TAKAGI,M; ZEBIB,A

    2003-09-01

    OAK-B135 Imperfections in PAMS mandrels critically govern the quality of final ICF targets. Imperfections in the mandrels can have a wide range of origins. Here, they present observations of 3 types of imperfections, and data to support the proposal that hydrodynamic factors during the curing of the mandrel are potential causes of these imperfections.

  11. 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

    Jayakar, Selwyn S; Zhou, Xiaojuan; Savechenkov, Pavel Y; Chiara, David C; Desai, Rooma; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B

    2015-09-18

    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-[(3)H]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-[(3)H]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.

  12. 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

  13. ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks

    PubMed Central

    Shen, Qiancheng; Wang, Guanqiao; Li, Shuai; Liu, Xinyi; Lu, Shaoyong; Chen, Zhongjie; Song, Kun; Yan, Junhao; Geng, Lv; Huang, Zhimin; Huang, Wenkang; Chen, Guoqiang; Zhang, Jian

    2016-01-01

    Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries. PMID:26365237

  14. Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

    PubMed Central

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J.; Walker, Laura M.; Ramos, Alejandra; Diwanji, Devan C.; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S.; Stanfield, Robyn L.; McBride, Ryan; Marozsan, Andre J.; Paulson, James C.; Sanders, Rogier W.; Moore, John P.; Burton, Dennis R.; Poignard, Pascal; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  15. Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the Brain.

    PubMed

    Orlandi, Cesare; Xie, Keqiang; Masuho, Ikuo; Fajardo-Serrano, Ana; Lujan, Rafael; Martemyanov, Kirill A

    2015-05-29

    Regulators of G protein signaling control the duration and extent of signaling via G protein-coupled receptor (GPCR) pathways by accelerating the GTP hydrolysis on G protein α subunits thereby promoting termination of GPCR signaling. A member of this family, RGS7, plays a critical role in the nervous system where it regulates multiple neurotransmitter GPCRs that mediate vision, memory, and the action of addictive drugs. Previous studies have established that in vivo RGS7 forms mutually exclusive complexes with the membrane protein RGS7-binding protein or the orphan receptor GPR158. In this study, we examine the impact of GPR158 on RGS7 in the brain. We report that knock-out of GPR158 in mice results in marked post-transcriptional destabilization of RGS7 and substantial loss of its association with membranes in several brain regions. We further identified the RGS7-binding site in the C terminus of GPR158 and found that it shares significant homology with the RGS7-binding protein. The proximal portion of the GPR158 C terminus additionally contained a conserved sequence that was capable of enhancing RGS7 GTPase-activating protein activity in solution by an allosteric mechanism acting in conjunction with the regulators of the G protein signaling-binding domain. The distal portion of the GPR158 C terminus contained several phosphodiesterase E γ-like motifs and selectively recruited G proteins in their activated state. The results of this study establish GPR158 as an essential regulator of RGS7 in the native nervous system with a critical role in controlling its expression, membrane localization, and catalytic activity.

  16. Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the Brain*♦

    PubMed Central

    Orlandi, Cesare; Xie, Keqiang; Masuho, Ikuo; Fajardo-Serrano, Ana; Lujan, Rafael; Martemyanov, Kirill A.

    2015-01-01

    Regulators of G protein signaling control the duration and extent of signaling via G protein-coupled receptor (GPCR) pathways by accelerating the GTP hydrolysis on G protein α subunits thereby promoting termination of GPCR signaling. A member of this family, RGS7, plays a critical role in the nervous system where it regulates multiple neurotransmitter GPCRs that mediate vision, memory, and the action of addictive drugs. Previous studies have established that in vivo RGS7 forms mutually exclusive complexes with the membrane protein RGS7-binding protein or the orphan receptor GPR158. In this study, we examine the impact of GPR158 on RGS7 in the brain. We report that knock-out of GPR158 in mice results in marked post-transcriptional destabilization of RGS7 and substantial loss of its association with membranes in several brain regions. We further identified the RGS7-binding site in the C terminus of GPR158 and found that it shares significant homology with the RGS7-binding protein. The proximal portion of the GPR158 C terminus additionally contained a conserved sequence that was capable of enhancing RGS7 GTPase-activating protein activity in solution by an allosteric mechanism acting in conjunction with the regulators of the G protein signaling-binding domain. The distal portion of the GPR158 C terminus contained several phosphodiesterase E γ-like motifs and selectively recruited G proteins in their activated state. The results of this study establish GPR158 as an essential regulator of RGS7 in the native nervous system with a critical role in controlling its expression, membrane localization, and catalytic activity. PMID:25792749

  17. An Analysis of the SLA-PAM Mailing List: What is SLA-PAM Doing?

    NASA Astrophysics Data System (ADS)

    Barve, S.; Dongare, S.

    2010-10-01

    The Physics-Astronomy-Mathematics (PAM) Division of the Special Libraries Association maintains an electronic discussion group which is referred to as PAMnet. In the present paper the PAM mailing list is analyzed from various points of view such as: different topics covered in this list, general nature of the mailing list, yearly postings, individual librarian's postings, postings from different countries, active participants, postings by publishers, postings on current topics, etc. The mailing list archive is available online from August 1998. It is an active mailing list where members get answers to their queries quickly. In the present paper, the mailing list is analyzed in detail to review trends in PAM libraries. A total number of 13 958 email messages (August 1998-September 2009) are analyzed to assess different trends.

  18. 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.

  19. 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.

  20. Allosteric proteins as logarithmic sensors

    PubMed Central

    Olsman, Noah; Goentoro, Lea

    2016-01-01

    Many sensory systems, from vision and hearing in animals to signal transduction in cells, respond to fold changes in signal relative to background. Responding to fold change requires that the system senses signal on a logarithmic scale, responding identically to a change in signal level from 1 to 3, or from 10 to 30. It is an ongoing search in the field to understand the ways in which a logarithmic sensor can be implemented at the molecular level. In this work, we present evidence that logarithmic sensing can be implemented with a single protein, by means of allosteric regulation. Specifically, we find that mathematical models show that allosteric proteins can respond to stimuli on a logarithmic scale. Next, we present evidence from measurements in the literature that some allosteric proteins do operate in a parameter regime that permits logarithmic sensing. Finally, we present examples suggesting that allosteric proteins are indeed used in this capacity: allosteric proteins play a prominent role in systems where fold-change detection has been proposed. This finding suggests a role as logarithmic sensors for the many allosteric proteins across diverse biological processes. PMID:27410043

  1. Pro-2-PAM Therapy for Central and Peripheral Cholinesterases

    PubMed Central

    DeMar, James C.; Clarkson, Edward D.; Ratcliffe, Ruthie H.; Campbell, Amy J.; Thangavelu, Sonia G.; Herdman, Christine A.; Leader, Haim; Schulz, Susan M.; Marek, Elizabeth; Medynets, Marie A.; Ku, Theresa C.; Evans, Sarah A.; Khan, Farhat A.; Owens, Roberta R.; Nambiar, Madhusoodana P.; Gordon, Richard K.

    2010-01-01

    Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980–1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using a) surgically-implanted radiotelemetry probes for electroencephalogram (EEG) b) neurohistopathology of brain, c) cholinesterase activities in the PNS and CNS, and d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropyl-fluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM, but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5 h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro 2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for

  2. 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

  3. 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

  4. Demonstration of the innate electrophilicity of 4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a small-molecule positive allosteric modulator of the glucagon-like peptide-1 receptor.

    PubMed

    Eng, Heather; Sharma, Raman; McDonald, Thomas S; Edmonds, David J; Fortin, Jean-Philippe; Li, Xianping; Stevens, Benjamin D; Griffith, David A; Limberakis, Chris; Nolte, Whitney M; Price, David A; Jackson, Margaret; Kalgutkar, Amit S

    2013-08-01

    4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R. PMID:23653442

  5. Allosteric modulation of an excitatory amino acid transporter: the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domain.

    PubMed

    Abrahamsen, Bjarke; Schneider, Nicole; Erichsen, Mette N; Huynh, Tri H V; Fahlke, Christoph; Bunch, Lennart; Jensen, Anders A

    2013-01-16

    In the present study, the mechanism of action and molecular basis for the activity of the first class of selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rodent ortholog GLAST are elucidated. The previously reported specificity of UCPH-101 and UCPH-102 for EAAT1 over EAAT2 and EAAT3 is demonstrated to extend to the EAAT4 and EAAT5 subtypes as well. Interestingly, brief exposure to UCPH-101 induces a long-lasting inactive state of EAAT1, whereas the inhibition exerted by closely related analogs is substantially more reversible in nature. In agreement with this, the kinetic properties of UCPH-101 unblocking of the transporter are considerably slower than those of UCPH-102. UCPH-101 exhibits noncompetitive inhibition of EAAT1, and its binding site in GLAST has been delineated in an elaborate mutagenesis study. Substitutions of several residues in TM3, TM4c, and TM7a of GLAST have detrimental effects on the inhibitory potency and/or efficacy of UCPH-101 while not affecting the pharmacological properties of (S)-glutamate or the competitive EAAT inhibitor TBOA significantly. Hence, UCPH-101 is proposed to target a predominantly hydrophobic crevice in the "trimerization domain" of the GLAST monomer, and the inhibitor is demonstrated to inhibit the uptake through the monomer that it binds to exclusively and not to affect substrate translocation through the other monomers in the GLAST trimer. The allosteric mode of UCPH-101 inhibition underlines the functional importance of the trimerization domain of the EAAT and demonstrates the feasibility of modulating transporter function through ligand binding to regions distant from its "transport domain." PMID:23325245

  6. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  7. 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.

  8. Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release.

    PubMed

    Foster, Daniel J; Wilson, Jermaine M; Remke, Daniel H; Mahmood, M Suhaib; Uddin, M Jashim; Wess, Jürgen; Patel, Sachin; Marnett, Lawrence J; Niswender, Colleen M; Jones, Carrie K; Xiang, Zixiu; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

    2016-09-21

    Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30 min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy. PMID:27618677

  9. Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

    PubMed Central

    Soukup, G A; DeRose, E C; Koizumi, M; Breaker, R R

    2001-01-01

    Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches. PMID:11345431

  10. 112 Gb/s transmission over 80 km SSMF using PDM-PAM4 and coherent detection without optical amplifier.

    PubMed

    Zhou, Xian; Zhong, Kangping; Huo, Jiahao; Gao, Lei; Wang, Yiguang; Wang, Liang; Yang, Yanfu; Yuan, Jinhui; Long, Keping; Zeng, Li; Lau, Alan Pak Tao; Lu, Chao

    2016-07-25

    Polarization-division-multiplexed (PDM) four-level pulse amplitude modulation (PAM4) with coherent detection is a promising low cost solution for 80 km inter-datacenter transmissions at 100 Gb/s and beyond. In this paper, three modified adaptive equalization algorithms for the PDM-PAM4 optical coherent systems, i.e. signal-phase aid least-mean-square (SP-LMS) algorithm, training multi-modulus algorithm (TMMA) and cascaded four-modulus algorithm (CMMA-4), are proposed and compared. Based on the proposed algorithms, 112 Gb/s PDM-PAM4 transmission over 80 km standard single mode fiber (SSMF) in C-band for a bit error rate (BER) below 3.8e-3 is successfully demonstrated without optical amplifier, chromatic dispersion (CD) pre-compensation and extra carrier recovery operations. PMID:27464183

  11. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA.

  12. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA. PMID:25908117

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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. PMID:25932491

  18. 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.

  19. 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.

  20. Survey on the PABC recognition motif PAM2.

    PubMed

    Albrecht, Mario; Lengauer, Thomas

    2004-03-26

    The PABP-interacting motif PAM2 has been identified in various eukaryotic proteins as an important binding site for the PABC domain. This domain is contained in homologs of the poly(A)-binding protein PABP and the ubiquitin-protein ligase HYD. Despite the importance of the PAM2 motif, a comprehensive analysis of its occurrence in different proteins has been missing. Using iterated sequence profile searches, we obtained an extensive list of proteins carrying the PAM2 motif. We discuss their functional context and domain architecture, which often consists of RNA-binding domains. Our list of PAM2 motif proteins includes eukaryotic homologs of eRF3/GSPT1/2, PAIP1/2, Tob1/2, Ataxin-2, RBP37, RBP1, Blackjack, HELZ, TPRD, USP10, ERD15, C1D4.14, and the viral protease P29. The identification of the PAM2 motif in as yet uncharacterized proteins can give valuable hints with respect to their cellular function and potential interaction partners and suggests further experimentation. It is also striking that the PAM2 motif appears to occur solely outside globular protein domains.

  1. Allosteric function and dysfunction of the prion protein.

    PubMed

    Linden, Rafael; Cordeiro, Yraima; Lima, Luis Mauricio T R

    2012-04-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.

  2. 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.

  3. Looking for the Origin of Allosteric Cooperativity in Metallopolymers.

    PubMed

    Babel, Lucille; Hoang, Thi Nhu Y; Guénée, Laure; Besnard, Céline; Wesolowski, Tomasz A; Humbert-Droz, Marie; Piguet, Claude

    2016-06-01

    The basic concept of allosteric cooperativity used in biology, chemistry and physics states that any change in the intermolecular host-guest interactions operating in multisite receptors can be assigned to intersite interactions. Using lanthanide metals as guests and linear multi-tridentate linear oligomers of variable lengths and geometries as hosts, this work shows that the quantitative modeling of metal loadings requires the consideration of a novel phenomenon originating from solvation processes. It stepwise modulates the intrinsic affinity of each isolated site in multisite receptors, and this without resorting to allosteric cooperativity. An easy-to-handle additive model predicts a negative power law dependence of the intrinsic affinity on the length of the linear metallopolymer. Applied to lanthanidopolymers, the latter common analysis overestimates cooperativity factors by more than two orders of magnitude. PMID:27142083

  4. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    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. PMID:27297398

  5. Acetylcholine Receptor: An Allosteric Protein

    NASA Astrophysics Data System (ADS)

    Changeux, Jean-Pierre; Devillers-Thiery, Anne; Chemouilli, Phillippe

    1984-09-01

    The nicotine receptor for the neurotransmitter acetylcholine is an allosteric protein composed of four different subunits assembled in a transmembrane pentamer α 2β γ δ . The protein carries two acetylcholine sites at the level of the α subunits and contains the ion channel. The complete sequence of the four subunits is known. The membrane-bound protein undergoes conformational transitions that regulate the opening of the ion channel and are affected by various categories of pharmacologically active ligands.

  6. 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

  7. PAMS photo image retrieval prototype system requirements specification

    SciTech Connect

    Conner, M.L.

    1996-04-30

    This project is part of the Photo Audiovisual Management System (PAMS). The project was initially identified in 1989 and has since been has been worked on under various names such as Image Retrieval and Viewing System, Photo Image Retrieval Subsystem and Image Processing and Compression System. This document builds upon the information collected and the analysis performed in the earlier phases of this project. The PAMS Photo Imaging subsystem will provide the means of capturing low resolution digital images from Photography`s negative files and associating the digital images with a record in the PAMS photo database. The digital images and key photo identification information will be accessible to HAN users to assist in locating and identifying specific photographs. After identifying desired photographs, users may request photo prints or high resolution digital images directly from Photography. The digital images captured by this project are for identification purposes only and are not intended to be of sufficient quality for subsequent use.

  8. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  9. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  10. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

    PubMed

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K; Zhu, Hu; Chen, Xin; Moy, Sheryl S; Saddoris, Kara A; Nikolova, Viktoriya D; 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

    2015-11-26

    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 the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 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

  11. 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

  12. 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.

  13. Pam17 and Tim44 act sequentially in protein import into the mitochondrial matrix.

    PubMed

    Schiller, Dirk

    2009-11-01

    Import of proteins into the matrix is driven by the Tim23 presequence translocase-associated import motor PAM. The core component of PAM is the mitochondrial chaperone mtHsp70, which ensures efficient translocation of proteins across the inner membrane through interactions with the J-protein complex Pam16-Pam18 (Tim16-Tim14) and its cochaperone Tim44. The recently identified non-essential Pam17 is a further member of PAM. Genetic and biochemical analyses reveal synthetic interactions between PAM17 and TIM44. Pam17 is involved in an early stage of protein translocation whereas Tim44 assists in a later step of transport, suggesting that both proteins can cooperate in a complementary manner in protein import.

  14. 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...

  15. 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...

  16. Polarization-insensitive PAM-4-carrying free-space orbital angular momentum (OAM) communications.

    PubMed

    Liu, Jun; Wang, Jian

    2016-02-22

    We present a simple configuration incorporating single polarization-sensitive phase-only liquid crystal spatial light modulator (SLM) to facilitate polarization-insensitive free-space optical communications employing orbital angular momentum (OAM) modes. We experimentally demonstrate several polarization-insensitive optical communication subsystems by propagating a single OAM mode, multicasting 4 and 10 OAM modes, and multiplexing 8 OAM modes, respectively. Free-space polarization-insensitive optical communication links using OAM modes that carry four-level pulse-amplitude modulation (PAM-4) signal are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties are less than 1 dB in both polarization-insensitive N-fold OAM modes multicasting and multiple OAM modes multiplexing at a bit-error rate (BER) of 2e-3 (enhanced forward-error correction (EFEC) threshold).

  17. 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.

  18. Polarization-insensitive PAM-4-carrying free-space orbital angular momentum (OAM) communications.

    PubMed

    Liu, Jun; Wang, Jian

    2016-02-22

    We present a simple configuration incorporating single polarization-sensitive phase-only liquid crystal spatial light modulator (SLM) to facilitate polarization-insensitive free-space optical communications employing orbital angular momentum (OAM) modes. We experimentally demonstrate several polarization-insensitive optical communication subsystems by propagating a single OAM mode, multicasting 4 and 10 OAM modes, and multiplexing 8 OAM modes, respectively. Free-space polarization-insensitive optical communication links using OAM modes that carry four-level pulse-amplitude modulation (PAM-4) signal are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties are less than 1 dB in both polarization-insensitive N-fold OAM modes multicasting and multiple OAM modes multiplexing at a bit-error rate (BER) of 2e-3 (enhanced forward-error correction (EFEC) threshold). PMID:26907073

  19. In vitro selection of allosteric ribozymes that sense the bacterial second messenger c-di-GMP.

    PubMed

    Furukawa, Kazuhiro; Gu, Hongzhou; Breaker, Ronald R

    2014-01-01

    Recently, a number of study have shown the ligand-dependent allosteric ribozymes can be harnessed as biosensors, high-throughput screening, and agents for the control of gene expression in vivo, called artificial riboswitches. In this chapter, we describe how in vitro selection can be used to create an allosteric ribozyme that senses bacterial second messenger cyclic-di-GMP (c-di-GMP). A hammerhead ribozyme was joined to a natural c-di-GMP class I riboswitch aptamer via communication modules. Both c-di-GMP-activating and -inhibiting ribozyme can be obtained by this approach. PMID:24549622

  20. Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

    PubMed

    Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

    2016-04-01

    CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. PMID:27041224

  1. 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

  2. Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter.

    PubMed

    Zhong, Huailing; Sánchez, Connie; Caron, Marc G

    2012-02-15

    The serotonin transporter (SERT) functions to transport serotonin (5-HT) from the extracellular space into neurons to maintain homeostatic control of 5-HT. It is the molecular target for selective serotonin reuptake inhibitor (SSRI) antidepressants. Preclinical research has shown that some SERT inhibitors can bind to two distinct binding sites on the SERT, a primary high affinity binding site and a low affinity allosteric binding site. Mutational studies of the SERT and computational modeling methods with escitalopram resulted in the identification of key amino acid residues important for the function of the allosteric binding site. While this allosteric binding site appears to influence the clinical efficacy of escitalopram under physiological conditions, the molecular mechanism of this effect is still poorly understood and may involve a large network of protein-protein interactions with the SERT. Dynamic interfaces between the SERT and the SERT interacting proteins (SIPs) potentially influence not only the SERT on its uptake function, its regulation, and trafficking, but also on known as well as yet to be identified non-canonical signaling pathways through SIPs. In this commentary, we outline approaches in the areas of selective small-molecule allosteric compound discovery, biochemistry, in vivo genetic knock-in mouse models, as well as computational and structural biology. These studies of the intra-molecular allosteric modulation of the SERT in the context of the myriad of potential inter-molecular signaling interactions with SIPs may help uncover unknown physiological functions of the SERT.

  3. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling

    PubMed Central

    Bock, Andreas; Merten, Nicole; Schrage, Ramona; Dallanoce, Clelia; Bätz, Julia; Klöckner, Jessica; Schmitz, Jens; Matera, Carlo; Simon, Katharina; Kebig, Anna; Peters, Lucas; Müller, Anke; Schrobang-Ley, Jasmin; Tränkle, Christian; Hoffmann, Carsten; De Amici, Marco; Holzgrabe, Ulrike; Kostenis, Evi; Mohr, Klaus

    2012-01-01

    Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling. PMID:22948826

  4. Performance evaluation of multilevel modulation formats using partial response for capacity upgrade in access network with limited electronic bandwidth

    NASA Astrophysics Data System (ADS)

    Madsen, Peter; Frejstrup Suhr, Lau; Sebastian Rodriguez, Juan; Tafur Monroy, Idelfonso; Vegas Olmos, Juan José

    2016-09-01

    We present a successful experimental evaluation of 4 level Pulse Amplitude Modulation (4-PAM) and Duobinary modulation. An experimental performance evaluation is presented for Duobinary 4 PAM and other modulation formats. All modulation formants used, may be considered to be implemented in future Passive Optical Network (PON) class access networks with limited electrical bandwidth. We compared NRZ, Duobinary, 4-PAM and Duobinary 4-PAM operating at 9 Gbaud over 20 km single mode fiber. The results provides an insight and guidelines on the utilization of these advanced modulation formats.

  5. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  6. 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.

  7. 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.

  8. 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

  9. Temperature-induced inversion of allosteric phenomena.

    PubMed

    Braxton, B L; Tlapak-Simmons, V L; Reinhart, G D

    1994-01-01

    Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation. In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand. Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate. The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner. These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography. PMID:8276837

  10. 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.

  11. First clinical trials of the Twente photoacoustic mammoscope (PAM)

    NASA Astrophysics Data System (ADS)

    Vaartjes, Susanne E.; van Hespen, Johan C. G.; Klaase, Joost M.; van den Engh, Frank M.; Thé, Andy K. H.; Steenbergen, Wiendelt; van Leeuwen, Ton G.; Manohar, Srirang

    2007-07-01

    Breast cancer dominates cancers in females. This burden on society and the room for improvements in the current practice of mammography have been stimuli for developing new modalities like photoacoustic mammography. At the University of Twente (UT), an instrument had been developed aimed at performing limited area scans on the human breast. This instrument is called the Twente Photoacoustic Mammoscope (PAM). The PAM is based on generating laserinduced ultrasound from absorbing structures in the breast. The heart of the instrument is a flat PVDF based detector matrix comprising 590 active elements. We show the performance characteristics of the ultrasound detector. The exciting source is an Nd:YAG laser operating at 1064 nm with 5 ns pulses. A study protocol was designed to explore the feasibility of using the PAM to detect cancer in the breasts of patients. The protocol was executed at the Medisch Spectrum Twente by using the mammoscope to obtain photoacoustic region-of-interest (ROI) images of the suspect/symptomatic breasts. We compare the photoacoustic images obtained with x-ray mammograms and ultrasound images. We show photoacoustic images of ROI in one case where we attribute high intensity regions to tumor vascularization.

  12. Assessment of subunit-dependent direct gating and allosteric modulatory effects of carisoprodol at GABA(A) receptors.

    PubMed

    Kumar, Manoj; González, Lorie A; Dillon, Glenn H

    2015-10-01

    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.

  13. 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.

  14. 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

  15. Pulse amplitude modulated chlorophyll fluorometer

    SciTech Connect

    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.

  16. 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

  17. 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.

  18. 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. PMID:26878552

  19. 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.

  20. The Role of PAM4 in the Management of Pancreatic Cancer: Diagnosis, Radioimmunodetection, and Radioimmunotherapy

    PubMed Central

    Han, Suxia; Jin, Guihua; Wang, Lijuan; Li, Meng; He, Chenchen; Guo, Xijing; Zhu, Qing

    2014-01-01

    PAM4, a new monoclonal antibody (MAb) known as clivatuzumab, is highly reactive with pancreatic cancer and precursor lesions. It is absent from the normal tissues and has limited reactivity with nonpancreatic cancer. The detailed characteristic of the PAM4 epitope is unknown but recent studies have shown that it is dependent on MUC1 glycosylation status. The limited PAM4 expression pattern makes it an attractive candidate for management of pancreatic adenocarcinoma. In addition, PAM4 is a serum biomarker for diagnosis of pancreatic cancer. Several different radiolabeled immunodiagnostic and immunotherapeutic agents of PAM4 have been developed and some are being evaluated in preclinical and/or clinical studies. The review will focus on PAM4 and its potential utility for the diagnosis, radioimmunodetection, and radioimmunotherapy of pancreatic cancer. PMID:24818166

  1. 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.

  2. 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

  3. 50th anniversary of the word "allosteric".

    PubMed

    Changeux, Jean-Pierre

    2011-07-01

    A brief historical account on the origin and meaning of the word "allosteric" is presented. The word was coined in an attempt to qualify the chemical mechanism of the feedback inhibition of bacterial enzymes by regulatory ligands. The data lead to the proposal that, at variance with the classical mechanism of mutual exclusion by steric hindrance, the inhibition takes place through an "allosteric" interaction between "no overlapping", stereospecifically distinct, sites for substrate and feedback inhibitor, mediated by a discrete reversible alteration of the molecular structure of the protein. PMID:21574197

  4. 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

  5. Laboratory Studies to Examine the Impact of Polyacrylamide (PAM) on Soil Hydraulic Conductivity

    NASA Astrophysics Data System (ADS)

    Moran, E. A.; Young, M. H.; Yu, Z.

    2005-12-01

    Polyacrylamide (PAM) is a long-chain synthetic polymer made of the monomer acrylamide (AMD). PAM has numerous uses ranging from food processing to drilling to wastewater treatment. More recently it has been proposed as a canal sealant in the western US to improve water conservation. To support a larger field-based experimental program being implemented in Grand Junction, CO, soil column experiments are being conducted to evaluate the mechanisms of how, and to what extent, PAM reduces soil hydraulic conductivity. The goal of the experiments is to find the optimum concentration and application method of PAM that reduces hydraulic conductivity to the greatest extent. Column tests were conducted, in triplicate, using a constant head method in acrylic columns of 15 cm length and 6.4 cm diameter. An unbalanced multi-factorial design was used with experimental variables including soil type (medium silica sand, locally-derived sand, and locally-derived loam), PAM concentration (11, 22, 44, 88 kg/canal-ha), turbidity (0, 100, 350 NTU), and application method (hydrated PAM on dry soil and powdered PAM applied to water column above saturated soil). Non-crosslinked anionic PAM with a molecular weight of 12 to 24 Mg/mol was used for all experiments. Additional experiments were conducted in graduated cylinders to evaluate interactions between PAM, turbidity and water chemistry. Results of the laboratory tests will be presented and discussed in the context of water conservation in the western US.

  6. 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.

  7. Detecting Allosteric Networks Using Molecular Dynamics Simulation.

    PubMed

    Bowerman, S; Wereszczynski, J

    2016-01-01

    Allosteric networks allow enzymes to transmit information and regulate their catalytic activities over vast distances. In principle, molecular dynamics (MD) simulations can be used to reveal the mechanisms that underlie this phenomenon; in practice, it can be difficult to discern allosteric signals from MD trajectories. Here, we describe how MD simulations can be analyzed to reveal correlated motions and allosteric networks, and provide an example of their use on the coagulation enzyme thrombin. Methods are discussed for calculating residue-pair correlations from atomic fluctuations and mutual information, which can be combined with contact information to identify allosteric networks and to dynamically cluster a system into highly correlated communities. In the case of thrombin, these methods show that binding of the antagonist hirugen significantly alters the enzyme's correlation landscape through a series of pathways between Exosite I and the catalytic core. Results suggest that hirugen binding curtails dynamic diversity and enforces stricter venues of influence, thus reducing the accessibility of thrombin to other molecules. PMID:27497176

  8. 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.

  9. An Allosteric Circuit in Caspase-1

    SciTech Connect

    Datta, D.; Scheer, J.M.; Romanowski, M.J.; Wells, J.A.

    2009-05-14

    Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface approximately 15 A from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them-Arg286 and Glu390, which form a salt bridge-have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k(cat)/K(m)). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k(cat)/K(m). X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or 'hot wire' that promotes site-to-site coupling.

  10. 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....

  11. 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....

  12. 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....

  13. 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....

  14. 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...

  15. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Virginia § 52.2426 Photochemical Assessment Monitoring Stations (PAMS) Program. On November 23, 1994 Virginia's... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all...

  16. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Virginia § 52.2426 Photochemical Assessment Monitoring Stations (PAMS) Program. On November 23, 1994 Virginia's... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all...

  17. 40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Virginia § 52.2426 Photochemical Assessment Monitoring Stations (PAMS) Program. On November 23, 1994 Virginia's... Stations (PAMS) Program on September 11, 1995 and made it part of the Virginia SIP. As with all...

  18. PHBV/PAM Scaffolds with Local Oriented Structure through UV Polymerization for Tissue Engineering

    PubMed Central

    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. PMID:24579074

  19. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1080 Photochemical Assessment Monitoring Stations (PAMS) Program. On March 24, 1994 Maryland's... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  20. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1080 Photochemical Assessment Monitoring Stations (PAMS) Program. On March 24, 1994 Maryland's... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  1. 40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1080 Photochemical Assessment Monitoring Stations (PAMS) Program. On March 24, 1994 Maryland's... (PAMS) Program on September 11, 1995 and made it part of Maryland SIP. As with all components of the...

  2. 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.

  3. 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.

  4. Anionic Polyacrylamide (PAM) and Extracellular Polysaccharides (EPS) effects on flocculation and aggregate stability of soil

    NASA Astrophysics Data System (ADS)

    Albalasmeh, A. A.; Gharaibeh, M. A.; Ghezzehei, T. A.

    2015-12-01

    Soil structure influences many soil properties including aeration, water retention, drainage, bulk density, and resistance to erosion and indirectly influences most biological and chemical processes that occur in and around soil. A significant amount of literature showed that PAM plays an important role to control erosion. However, researchers are looking for more natural alternative for PAM. This study evaluated two anionic polymers including low and high molecular weight (MW), root exudates and bacterial exudates. We evaluated their influence on the rate and efficacy of colloid flocculation and the percent of water stable aggregates. We found that PAM was more effective than EPS in flocculating the colloids and all polymers increased the percent of stable soil aggregates although the PAM was more effective. These data suggest that the EPS would be less effective than PAM for reducing water erosion owing to its lesser flocculation and aggregate stabilizing potential.

  5. Neuronal activity patterns in the mediodorsal thalamus and related cognitive circuits are modulated by metabotropic glutamate receptors

    PubMed Central

    Copeland, C.S.; Neale, S.A.; Salt, T.E.

    2015-01-01

    The mediodorsal thalamus (MD) likely plays an important role in cognition as it receives abundant afferent connections from the amygdala and prefrontal cortex (PFC). Indeed, disturbed activity within the MD is thought to precipitate cognitive deficits associated with schizophrenia. As compounds acting at the Group II metabotropic glutamate (mGlu) receptors (subtypes mGlu2/mGlu3) have efficacy in animal models of schizophrenia, we investigated whether a Group II agonist and an mGlu2 positive allosteric modulator (PAM) could modulate MD activity. Extracellular single-unit recordings were made in vivo from MD neurones in anaesthetised rats. Responses were elicited by electrical stimulation of the PFC and/or amygdala, with Group II compounds locally applied as required. The Group II agonist reduced inhibition evoked in the MD: an effect manifested as an increase in short-latency responses, and a decrease in long-latency burst-firing. This disinhibitory action of the Group II receptors in the MD represents a mechanism of potential therapeutic importance as increased inhibition in the MD has been associated with cognitive deficit-onset. Furthermore, as co-application of the mGlu2 PAM did not potentiate the Group II agonist effects in the MD, we suggest that the Group II disinhibitory effect is majority-mediated via mGlu3. This heterogeneity in Group II receptor thalamic physiology bears consequence, as compounds active exclusively at the mGlu2 subtype are unlikely to perturb maladapted MD firing patterns associated with cognitive deficits, with activity at mGlu3 receptors possibly more appropriate. Indeed, polymorphisms in the mGlu3, but not the mGlu2, gene have been detected in patients with schizophrenia. PMID:25576798

  6. Neuronal activity patterns in the mediodorsal thalamus and related cognitive circuits are modulated by metabotropic glutamate receptors.

    PubMed

    Copeland, C S; Neale, S A; Salt, T E

    2015-05-01

    The mediodorsal thalamus (MD) likely plays an important role in cognition as it receives abundant afferent connections from the amygdala and prefrontal cortex (PFC). Indeed, disturbed activity within the MD is thought to precipitate cognitive deficits associated with schizophrenia. As compounds acting at the Group II metabotropic glutamate (mGlu) receptors (subtypes mGlu2/mGlu3) have efficacy in animal models of schizophrenia, we investigated whether a Group II agonist and an mGlu2 positive allosteric modulator (PAM) could modulate MD activity. Extracellular single-unit recordings were made in vivo from MD neurones in anaesthetised rats. Responses were elicited by electrical stimulation of the PFC and/or amygdala, with Group II compounds locally applied as required. The Group II agonist reduced inhibition evoked in the MD: an effect manifested as an increase in short-latency responses, and a decrease in long-latency burst-firing. This disinhibitory action of the Group II receptors in the MD represents a mechanism of potential therapeutic importance as increased inhibition in the MD has been associated with cognitive deficit-onset. Furthermore, as co-application of the mGlu2 PAM did not potentiate the Group II agonist effects in the MD, we suggest that the Group II disinhibitory effect is majority-mediated via mGlu3. This heterogeneity in Group II receptor thalamic physiology bears consequence, as compounds active exclusively at the mGlu2 subtype are unlikely to perturb maladapted MD firing patterns associated with cognitive deficits, with activity at mGlu3 receptors possibly more appropriate. Indeed, polymorphisms in the mGlu3, but not the mGlu2, gene have been detected in patients with schizophrenia. PMID:25576798

  7. Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.

    PubMed

    McGresham, Maria S; Reinhart, Gregory D

    2015-01-27

    The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol(-1) (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol(-1) increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild

  8. Preferential Acquisition and Activation of Plasminogen Glycoform II by PAM Positive Group A Streptococcal Isolates.

    PubMed

    De Oliveira, David M P; Law, Ruby H P; Ly, Diane; Cook, Simon M; Quek, Adam J; McArthur, Jason D; Whisstock, James C; Sanderson-Smith, Martina L

    2015-06-30

    Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289. PMID:26029848

  9. Time domain zero-padding based adaptive-PAM signal transmission with high spectral efficiency in IMDD optical communication system

    NASA Astrophysics Data System (ADS)

    Zhang, Fangliu; He, Jing; Deng, Rui; Cheng, Yun; Xiao, Minlei; Chen, Lin

    2016-08-01

    In this paper, an adaptive pulse amplitude modulation (APAM) scheme is proposed and experimentally demonstrated in the intensity-modulation and direct-detection (IMDD) optical communications system. In the proposed scheme, the channel is divided into two sub-channels, and different PAM mapping can be chosen for different sub-channel according to the fading conditions. In addition, the 20-km standard single mode fiber (SSMF) transmission of 24 Gbit/s 16/4-APAM signal with the spectral efficiency (SE) up to 6 bit/s/Hz is experimentally demonstrated. The experiment results show that the bit error rate (BER) of the 16/4-APAM signal can be achieved less than 2.4e-2.

  10. Design of an allosterically regulated retroaldolase

    PubMed Central

    Raymond, Elizabeth A; Mack, Korrie L; Yoon, Jennifer H; Moroz, Olesia V; Moroz, Yurii S; Korendovych, Ivan V

    2015-01-01

    We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca2+ ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue. PMID:25516403

  11. Molecular Basis of Allosteric Transitions: GroEL

    NASA Astrophysics Data System (ADS)

    Horovitz, Amnon

    Chaperonins such as GroEL from Escherichia coli are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. Here, we describe some of the various functional (allosteric) states of GroEL, the pathways by which they inter-convert and the coupling between allosteric transitions and protein folding reactions.

  12. Universal allosteric mechanism for Gα activation by GPCRs

    PubMed Central

    Flock, Tilman; Venkatakrishnan, A. J.; Kayikci, Melis; Tate, Christopher G.; Veprintsev, Dmitry B.; Babu, M. Madan

    2016-01-01

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ~800 human GPCRs and 16 different Gα proteins, does a universal allosteric mechanism govern Gα activation? Here we show that different GPCRs interact and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that can undergo disorder-order transitions decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, whilst conserving the allosteric activation mechanism. PMID:26147082

  13. Universal allosteric mechanism for Gα activation by GPCRs.

    PubMed

    Flock, Tilman; Ravarani, Charles N J; Sun, Dawei; Venkatakrishnan, A J; Kayikci, Melis; Tate, Christopher G; Veprintsev, Dmitry B; Babu, M Madan

    2015-08-13

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism. PMID:26147082

  14. The global allostery model of hemoglobin: an allosteric mechanism involving homotropic and heterotropic interactions.

    PubMed

    Yonetani, Takashi; Tsuneshige, Antonio

    2003-06-01

    Studies of the allosteric mechanism of hemoglobin (Hb) have evolved from phenomenological descriptions to structure-based molecular mechanisms, as the molecular structures of Hb in deoxy and ligated states have been elucidated. The MWC two-state concerted model has been the widely accepted as the most plausible of the allosteric mechanisms of Hb. It assumes that the O2-affinity of Hb is regulated/controlled primarily by the T/R quaternary structural transition and that heterotropic effectors bind preferentially to T (deoxy) Hb to shift the T/R allosteric equilibrium toward the T state. However, recent more comprehensive O2-binding measurements of Hb have revealed a new mechanism, the Global Allostery model. It describes that the O2-affinity and the cooperativity are modulated in greater extents and the Bohr effect is generated primarily by the tertiary structural changes in both T (deoxy) and R (ligated) states of Hb. Differential interactions of heterotropic allosteric effectors with both T (deoxy) and R (ligated) states of Hb induce these tertiary structural changes. The X-ray structure of a complex of R (ligated) Hb with BZF, a potent heterotropic effector, has revealed the stereo-chemical influence of these effectors on the structure of R (ligated) Hb, resulting in the reduction of the ligand affinity in R (ligated) Hb. This model stresses the fundamental importance of the heterotropic interactions in regulation/control of the functionality of Hb. They alter the tertiary structures of both T (deoxy) and R (oxy) Hb, leading to large-scale modulations of the O2 affinity (KT and KR), and consequently the cooperativity (KR/KT) and the Bohr effect (delta P50/delta pH) from a global viewpoint of allostery in Hb.

  15. Wind tunnel experimental study on the effect of PAM on soil wind erosion control.

    PubMed

    He, Ji-Jun; Cai, Qiang-Guo; Tang, Ze-Jun

    2008-10-01

    In recent years, high-molecular-weight anionic polyacrylamide (PAM) have been widely tested on a variety of soils, primarily in water erosion control. However, little information is available regarding the effectiveness of PAM on preventing soil loss from wind erosion. The research adopted room wind tunnel experiment, two kinds of soils were used which were from the agro-pastoral area of Inner Mongolia, the northwest of China, the clay content of soils were 22.0 and 13.7%, respectively. For these tests, all the treatments were performed under the condition of wind velocity of 14 m s(-1) and a blown angle of 8.75%, according to the actual situation of experimented area. The study results indicated that using PAM on the soil surface could enhance the capability of avoiding the wind erosion, at the same time, the effect of controlling wind soil erosion with 4 g m(-2) PAM was better than 2 g m(-2) PAM's. Economically, the 2 g m(-2) PAM used in soil surface can control wind erosion effectively in this region. The prophase PAM accumulated in soil could not improve the capability of avoiding the wind erosion, owing to the degradation of PAM in the soil and the continual tillage year after year. The texture of soil is a main factor influencing the capability of soil avoiding wind erosion. Soil with higher clay content has the higher capability of preventing soil from wind erosion than one with the opposite one under the together action of PAM and water.

  16. Multi-parametric relationships between PAM measurements and carbon incorporation, an in situ approach.

    PubMed

    Napoléon, Camille; Claquin, Pascal

    2012-01-01

    Primary production (PP) in the English Channel was measured using (13)C uptake and compared to the electron transport rate (ETR) measured using PAM (pulse amplitude modulated fluorometer). The relationship between carbon incorporation (P(obs)) and ETR was not linear but logarithmic. This result can be explained by alternative electron sinks at high irradiance which protect the phytoplankton from photoinhibition. A multi-parametric model was developed to estimate PP by ETR. This approach highlighted the importance of taking physicochemical parameters like incident light and nutrient concentrations into account. The variation in the ETR/P(obs) ratio as a function of the light revealed different trends which were characterized by three parameters (R(max), the maximum value of ETR/P(obs); E(Rmax), the light intensity at which R(max) is measured; γ the initial slope of the curve). Based on the values of these three parameters, data were divided into six groups which were highly dependent on the seasons and on the physicochemical conditions. Using the multi-parametric model which we defined by P(obs) and ETR measurements at low frequencies, the high frequency measurements of ETR enabled us to estimate the primary production capacity between November 2009 and December 2010 at high temporal and spatial scales.

  17. Multi-Parametric Relationships between PAM Measurements and Carbon Incorporation, an In Situ Approach

    PubMed Central

    Napoléon, Camille; Claquin, Pascal

    2012-01-01

    Primary production (PP) in the English Channel was measured using 13C uptake and compared to the electron transport rate (ETR) measured using PAM (pulse amplitude modulated fluorometer). The relationship between carbon incorporation (Pobs) and ETR was not linear but logarithmic. This result can be explained by alternative electron sinks at high irradiance which protect the phytoplankton from photoinhibition. A multi-parametric model was developed to estimate PP by ETR. This approach highlighted the importance of taking physicochemical parameters like incident light and nutrient concentrations into account. The variation in the ETR/Pobs ratio as a function of the light revealed different trends which were characterized by three parameters (Rmax, the maximum value of ETR/Pobs; ERmax, the light intensity at which Rmax is measured; γ the initial slope of the curve). Based on the values of these three parameters, data were divided into six groups which were highly dependent on the seasons and on the physicochemical conditions. Using the multi-parametric model which we defined by Pobs and ETR measurements at low frequencies, the high frequency measurements of ETR enabled us to estimate the primary production capacity between November 2009 and December 2010 at high temporal and spatial scales. PMID:22911698

  18. An improved recurrent neural network for M-PAM symbol detection.

    PubMed

    Hacioglu, K

    1997-01-01

    In this paper, a fully connected recurrent neural network (RNN) is presented for the recovery of M-ary pulse amplitude modulated (M-PAM) signals in the presence of intersymbol interference and additive white Gaussian noise. The network makes use of two different activation functions. One is the traditional two-level sigmoid function, which is used at its hidden nodes, and the other is the M-level sigmoid function (MSF), which is used at the output node. The shape of the M-level activation function is controlled by two parameters: the slope and shifting parameters. The effect of these parameters on the learning performance is investigated through extensive simulations. In addition, the network is compared with a linear transversal equalizer, a decision feedback equalizer and a recently proposed RNN equalizer which has used a scaled sigmoid function (SSF) at its output node. Comparisons are made in terms of their learning properties and symbol error rates. It is demonstrated that the proposed RNN equalizer performs better, provided that the MSF parameters are properly selected. PMID:18255677

  19. Generation 3 programmable array microscope (PAM) for high speed large format optical sectioning in fluorescence

    NASA Astrophysics Data System (ADS)

    de Vries, Anthony H. B.; Cook, Nathan P.; Kramer, Stephan; Arndt-Jovin, Donna J.; Jovin, Thomas M.

    2015-03-01

    We report on the current version of the optical sectioning programmable array microscope (PAM) implemented with a single digital micro-mirror device (DMD) spatial light modulator utilized as a mask in both the fluorescence excitation and emission paths. The PAM incorporates structured illumination and structured detection operating in synchrony. A sequence of binary patterns of excitation light in high definition format (1920×1080 elements) is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the Texas Instruments 1080p DMD. The resulting fluorescent emission is captured as two distinct signals: conjugate (c, ca. "on-focus") consisting of light impinging on and deviated from the "on" elements of the DMD, and the non-conjugate (nc, ca. "out-of-focus") light falling on and deviated from the "off" elements. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The widefield image is given by the sum of the c and nc images. This procedure allows a high duty cycle (typically 25-50%) of on-elements in the excitation patterns and thus functions with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The corresponding acquisition speed is also very high, limited only by the bandwidth of the camera(s) (100 fps full frame with the sCMOS camera in current use) and the optical power of the light source (lasers, large area LEDs). In contrast to the static patterns typical of SIM systems, the programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, ranging from patterned

  20. Moving Beyond Active-Site Detection: MixMD Applied to Allosteric Systems.

    PubMed

    Ghanakota, Phani; Carlson, Heather A

    2016-08-25

    Mixed-solvent molecular dynamics (MixMD) is a hotspot-mapping technique that relies on molecular dynamics simulations of proteins in binary solvent mixtures. Previous work on MixMD has established the technique's effectiveness in capturing binding sites of small organic compounds. In this work, we show that MixMD can identify both competitive and allosteric sites on proteins. The MixMD approach embraces full protein flexibility and allows competition between solvent probes and water. Sites preferentially mapped by probe molecules are more likely to be binding hotspots. There are two important requirements for the identification of ligand-binding hotspots: (1) hotspots must be mapped at very high signal-to-noise ratio and (2) the hotspots must be mapped by multiple probe types. We have developed our mapping protocol around acetonitrile, isopropanol, and pyrimidine as probe solvents because they allowed us to capture hydrophilic, hydrophobic, hydrogen-bonding, and aromatic interactions. Charged probes were needed for mapping one target, and we introduce them in this work. In order to demonstrate the robust nature and wide applicability of the technique, a combined total of 5 μs of MixMD was applied across several protein targets known to exhibit allosteric modulation. Most notably, all the protein crystal structures used to initiate our simulations had no allosteric ligands bound, so there was no preorganization of the sites to predispose the simulations to find the allosteric hotspots. The protein test cases were ABL Kinase, Androgen Receptor, CHK1 Kinase, Glucokinase, PDK1 Kinase, Farnesyl Pyrophosphate Synthase, and Protein-Tyrosine Phosphatase 1B. The success of the technique is demonstrated by the fact that the top-four sites solely map the competitive and allosteric sites. Lower-ranked sites consistently map other biologically relevant sites, multimerization interfaces, or crystal-packing interfaces. Lastly, we highlight the importance of including protein

  1. 40 CFR 52.2035 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (PAMS) Program as a state implementation plan (SIP) revision, as required by section 182(c)(1) of the... 11, 1995 and made it part of Pennsylvania SIP. As with all components of the SIP, Pennsylvania...

  2. 40 CFR 52.2035 - Photochemical Assessment Monitoring Stations (PAMS) Program.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (PAMS) Program as a state implementation plan (SIP) revision, as required by section 182(c)(1) of the... 11, 1995 and made it part of Pennsylvania SIP. As with all components of the SIP, Pennsylvania...

  3. Three years' experience with the offshore self-boring pressuremeter ''PAM''

    SciTech Connect

    Brucy, F.; Fay, J.B.; LeTirant, P.

    1984-05-01

    The Offshore Self-Boring Pressuremeter (PAM) has been developed by the Institut Francais du Petrole for geotechnical investigations in water depths up to 1000 meters. The PAM was first presented at the 1981 OTC. It mainly consists of a seabed frame from which a self-boring pressuremeter probe is operated. To date, the PAM has totaled nearly 450 meters of borehole depth and more than 200 pressuremeter tests. Investigations have been performed in a wide variety of soil types. The latest deepwater campaign was carried out successfully in the Mediterranean at a water depth of 625 meters. Extensive geotechnical information is obtained from a PAM boring operation. The use of the pressuremeter method for offshore investigations and foundation designing should open up very interesting prospects.

  4. Allosteric inhibition of g-protein coupled receptor oligomerization: strategies and challenges for drug development.

    PubMed

    Hurevich, Mattan; Talhami, Alaa; Shalev, Deborah E; Gilon, Chaim

    2014-01-01

    G-protein coupled receptors (GPCRs) mediate a large number of biological pathways and are major therapeutic targets. One of the most exiting phenomena of GPCRs is their ability to interact with other GPCRs. GPCRGPCR interactions, also known as GPCR oligomerization, may create various functional entities such as homo- and heterodimers and also form complex multimeric GPCR clusters. In many biological systems, GPCR-GPCR interactions are crucial for signal regulation. The interaction with other receptors results in allosteric modifications of GPCRs through conformational changes. Allosteric inhibition of GPCRs is considered an attractive strategy for drug development and does not involve targeting the orthosteric site. Understanding the nature of GPCR-GPCR interactions is mandatory for developing allosteric inhibitors. Studying GPCR-GPCR interactions is a challenging task and many methods have been developed to analyze these events. This review will highlight some of the methods developed to study GPCR-GPCR interactions and will describe pivotal studies that provided the basic understanding of the importance of GPCR oligomerization. We will also describe the significance of GPCR interaction networks for drug development. Recent studies will be reviewed to illustrate the use of state-of-the-art biophysical and spectroscopic methods for the discovery of GPCR oligomerization modulators.

  5. 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

  6. Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase.

    PubMed

    Mathieu, Valéry; Fastrez, Jacques; Soumillion, Patrice

    2010-09-01

    In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.

  7. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    PubMed

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  8. Preparation and characterization of BC/PAM-AgNPs nanocomposites for antibacterial applications.

    PubMed

    Yang, Guang; Wang, Caixia; Hong, Feng; Yang, Xuexia; Cao, Zhangjun

    2015-01-22

    In this work, a bacterial cellulose/polyacrylamide (BC/PAM) double network composite was prepared to act as the template for in situ synthesis of silver nanoparticles (AgNPs). Effects of reaction conditions of the BC/PAM composite were investigated on its microstructure, mechanical properties and thermal stabilities. Both the BC/PAM composite and pure BC were utilized to prepare the corresponding silver impregnated nanocomposites, i.e., BC/PAM-AgNPs and BC-AgNPs, by an environmental friendly method, UV irradiation. The influences of the templates were investigated on the AgNPs formation and the antibacterial activities of the nanocomposites by both the zone of inhibition and dynamic shake flask methods. It was shown that the BC/PAM composite displayed a denser microstructure and higher thermal stabilities than pure BC. The BC/PAM-AgNPs nanocomposite exhibited a bigger particle size and lower mass content of AgNPs than the BC-AgNPs one. For the antibacterial test, two nanocomposites exhibited a close antibacterial effect, with a high log reduction above 3 and killing ratio above 99.9%, respectively. PMID:25439942

  9. Preparation and characterization of BC/PAM-AgNPs nanocomposites for antibacterial applications.

    PubMed

    Yang, Guang; Wang, Caixia; Hong, Feng; Yang, Xuexia; Cao, Zhangjun

    2015-01-22

    In this work, a bacterial cellulose/polyacrylamide (BC/PAM) double network composite was prepared to act as the template for in situ synthesis of silver nanoparticles (AgNPs). Effects of reaction conditions of the BC/PAM composite were investigated on its microstructure, mechanical properties and thermal stabilities. Both the BC/PAM composite and pure BC were utilized to prepare the corresponding silver impregnated nanocomposites, i.e., BC/PAM-AgNPs and BC-AgNPs, by an environmental friendly method, UV irradiation. The influences of the templates were investigated on the AgNPs formation and the antibacterial activities of the nanocomposites by both the zone of inhibition and dynamic shake flask methods. It was shown that the BC/PAM composite displayed a denser microstructure and higher thermal stabilities than pure BC. The BC/PAM-AgNPs nanocomposite exhibited a bigger particle size and lower mass content of AgNPs than the BC-AgNPs one. For the antibacterial test, two nanocomposites exhibited a close antibacterial effect, with a high log reduction above 3 and killing ratio above 99.9%, respectively.

  10. Viewpoint: Discriminating between noncompetitive and allosteric interactions.

    PubMed

    Ochs, Raymond S; Ashby, Charles R

    2008-03-01

    Understanding inhibition modes other than competitive is of clear importance in neuropharmacology. However, there appears to be some confusion concerning modes of inhibition that are not of the competitive type. It is critical to make a distinction between "not competitive" and "noncompetitive," as the later is a particular mode of inhibition. Further, there appears to be confusion between noncompetitive and allosteric behavior. Our purpose in this contribution is to explore the basis of these terms so that insight into pharmacological systems has a firmer mechanistic basis.

  11. A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

    PubMed Central

    Sineva, Elena V.; Rumfeldt, Jessica A. O.; Halpert, James R.; Davydov, Dmitri R.

    2013-01-01

    Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located. PMID:24376769

  12. Allosteric dominance in carbamoyl phosphate synthetase.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1999-02-01

    A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed. PMID:9931004

  13. Functional anatomy of an allosteric protein

    NASA Astrophysics Data System (ADS)

    Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

    2013-12-01

    Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

  14. Comparison of 2-PAM and pro-2-PAM containing treatment regimens as antagonists of nerve agent-induced lethality and incapacitation. Final report, June 1981-December 1985

    SciTech Connect

    Talbot, B.G.; Harris, L.W.; Lennox, W.J.; Anderson, D.A.; Green, M.D.

    1986-09-01

    In vivo, (2-Puridine Aldoxine Methioidide) reactivates phosphonylated acetylcholinesterase AChE peripherally, but is effective in restoring AChE centrally because the quaternary nitrogen atom of 2-PAM prevents penetration of the brain. The problem was solved by the synthesis of the 1,6-dihyropyridine derivative of 2-PAM, pro-2-PAM (PP). Functional brain AChE is related to return to control performance on an accelerating rotarod (ARR) in animals intoxicated with soman. There should be a difference in the time to recovery of control ARR performance between PP- and 2-PAM-treated, sarin-intoxicated animals. In the present work, an ARR decrement free dosage (DFD) of each of these oximes (30 mg/kg, im) in combination with DFD of atropine (A) and mecamylamine (M) (0.79 mg/kg each, im) was used as pretreatment against sarin-induced deficit. The same antidotes were given pre-and post- intoxication (as pretreatment and therapy) to anatagonize sarin-induced lethality; the PP containing antidote provided significantly greater protection than that by the 2-PAM antidote which in turn provided significant protection over control. Neither antidote when given as pretreatment and therapy provided protection above control against soman-induced physical incapacitation, but they were equally effective in antagonizing VX-induced physical incapacitation. The reversal of sarin-induced physical debilitation reflects the central actions of PP and supports the notion that functional brain AChE activity is essential for rapid recovery from the debilitating effeclts on nerve agents.

  15. 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

  16. 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.

  17. Skylab IMSS checklist application study for emergency medical care. [emergency medical care operations involving the use and operation of the portable ambulance module

    NASA Technical Reports Server (NTRS)

    Carl, J. G.; Furukawa, S.

    1975-01-01

    A manual is presented that provides basic technical documentation to support the operation and utilization of the Portable Ambulance Module (PAM) in the field. The PAM is designed to be used for emergency resuscitation and victim monitoring. The functions of all the controls, displays, and stowed equipment of the unit are defined. Supportive medical and physiological data in those areas directly related to the uses of the PAM unit are presented.

  18. PAM50 proliferation score as a predictor of weekly paclitaxel benefit in breast cancer.

    PubMed

    Martín, Miguel; Prat, Aleix; Rodríguez-Lescure, Alvaro; Caballero, Rosalía; Ebbert, Mark T W; Munárriz, Blanca; Ruiz-Borrego, Manuel; Bastien, Roy R L; Crespo, Carmen; Davis, Carole; Rodríguez, César A; López-Vega, José M; Furió, Vicente; García, Ana M; Casas, Maribel; Ellis, Matthew J; Berry, Donald A; Pitcher, Brandelyn N; Harris, Lyndsay; Ruiz, Amparo; Winer, Eric; Hudis, Clifford; Stijleman, Inge J; Tuck, David P; Carrasco, Eva; Perou, Charles M; Bernard, Philip S

    2013-04-01

    To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p < 0.001; and interaction test, p = 0.006). No significant interactions between treatment and the PAM50 subtypes or the various clinical-pathological variables, including Ki-67 and histologic grade, were identified. Finally, similar OS results were obtained in the CALGB data set, although the interaction test did not reach statistical significance (p = 0.109). The PAM50 proliferation score identifies a subset of patients with a low proliferation status that may derive a larger benefit from weekly paclitaxel.

  19. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

    PubMed

    Laprairie, Robert B; Kulkarni, Abhijit R; Kulkarni, Pushkar M; Hurst, Dow P; Lynch, Diane; Reggio, Patricia H; Janero, David R; Pertwee, Roger G; Stevenson, Lesley A; Kelly, Melanie E M; Denovan-Wright, Eileen M; Thakur, Ganesh A

    2016-06-15

    One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse

  20. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

    PubMed Central

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

    2015-01-01

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

  1. An allosteric model for the functional plasticity of olfactory chemoreceptors

    NASA Astrophysics Data System (ADS)

    Colosimo, Alfredo

    2000-12-01

    A simple allosteric model may describe the relatively (a)specific behaviour of olfactory chemoreceptors (OCs) and their functional plasticity with a minimum number of parameters. Allosteric, heterotropic effectors are suggested as a possible cause of variable responses documented, in particular, in frog OCs. As an immediate spinoff of the continuously increasing amount of structural information available on natural OCs, development of appropriate allosteric models is foreseen to provide plausible molecular mechanisms for their complex functional performance. This may also have implications in the design of artificial olfaction systems.

  2. Novel positive allosteric modulators of GABAA receptors with anesthetic activity

    PubMed Central

    Maldifassi, Maria C.; Baur, Roland; Pierce, David; Nourmahnad, Anahita; Forman, Stuart A.; Sigel, Erwin

    2016-01-01

    GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics. PMID:27198062

  3. Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

    SciTech Connect

    Ascenzi, Paolo; Ciaccio, Chiara; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo

    2011-01-07

    Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

  4. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    PubMed

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. PMID:26322498

  5. Broadening Staphylococcus aureus Cas9 Targeting Range by Modifying PAM Recognition

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu T.; Topkar, Ved V.; Zheng, Zongli; Joung, J. Keith

    2015-01-01

    CRISPR-Cas9 nucleases are primarily guided by RNA-DNA interactions but also require Cas9-mediated recognition of a protospacer adjacent motif (PAM). While potentially advantageous for specificity, extended PAM sequences limit the targeting range of Cas9 orthologues for genome editing. One possible strategy to relieve this restriction is to relax specificities for certain positions within the PAM. Here we used molecular evolution to modify the NNGRRT PAM specificity of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, shows robust genome editing activities at endogenous human target sites with NNNRRT PAMs. Importantly, using GUIDE-seq, we show that both wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. KKH SaCas9 increased the targeting range of SaCas9 by nearly two- to four-fold. Our molecular evolution strategy does not require structural information and therefore should be applicable to a wide range of Cas9 orthologues. PMID:26524662

  6. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    PubMed

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.

  7. Feasibility investigation of oily wastewater treatment by combination of zinc and PAM in coagulation/flocculation.

    PubMed

    Zeng, Yubin; Yang, Changzhu; Zhang, Jingdong; Pu, Wenhong

    2007-08-25

    Poly-zinc silicate (PZSS) is a new type of coagulant with cationic polymer synthesized by polysilicic acid and zinc sulfate. It has been used in several sorts of wastewaters treatment, but not used in oily wastewater treatment. In this study, we investigated the coagulation/flocculation of oil and suspended solids in heavy oil wastewater (HOW) by PZSS and anion polyacrylamide (A-PAM). The properties of PZSS cooperated with A-PAM were compared with PAC and PFS in dosages, PAMs amount, settling time, pH value and flocs morphology. The results showed that PZSS was more efficient than PAC and PFS. Under the optimum experimental conditions of coagulation/flocculation (dosage: 100mg/L, A-PAM dosage: 1.0mg/L, settling time time: 40min and pH 6.5-9.5), more than 99% of oil was removed and suspended solid value less than 5mg/L by using PZSS cooperated with A-PAM, which could satisfy the demands of the pre-treatment process for HOW to be reused in the steam boiler or recycled into the injecting well.

  8. Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum-Pressure Accelerated Movement (V-PAM).

    PubMed

    Yu, Zeta Tak For; Cheung, Mei Ki; Liu, Shirley Xiaosu; Fu, Jianping

    2016-09-01

    Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum-Pressure Accelerated Movement (V-PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead-end channels and closed chambers. Operation of the V-PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas-permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V-PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V-PAM for rapid filling of temperature-sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V-PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs-on-chips, and biomimetic studies. PMID:27409528

  9. Small-world networks of residue interactions in the Abl kinase complexes with cancer drugs: topology of allosteric communication pathways can determine drug resistance effects.

    PubMed

    Tse, A; Verkhivker, G M

    2015-07-01

    The human protein kinases play a fundamental regulatory role in orchestrating functional processes in complex cellular networks. Understanding how conformational equilibrium between functional kinase states can be modulated by ligand binding or mutations is critical for quantifying molecular basis of allosteric regulation and drug resistance. In this work, molecular dynamics simulations of the Abl kinase complexes with cancer drugs (Imatinib and Dasatinib) were combined with structure-based network modeling to characterize dynamics of the residue interaction networks in these systems. The results have demonstrated that structural architecture of kinase complexes can produce a small-world topology of the interaction networks. Our data have indicated that specific Imatinib binding to a small number of highly connected residues could lead to network-bridging effects and allow for efficient allosteric communication, which is mediated by a dominant pathway sensitive to the unphosphorylated Abl state. In contrast, Dasatinib binding to the active kinase form may activate a broader ensemble of allosteric pathways that are less dependent on the phosphorylation status of Abl and provide a better balance between the efficiency and resilience of signaling routes. Our results have unveiled how differences in the residue interaction networks and allosteric communications of the Abl kinase complexes can be directly related to drug resistance effects. This study offers a plausible perspective on how efficiency and robustness of the residue interaction networks and allosteric pathways in kinase structures may be associated with protein responses to drug binding.

  10. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

    PubMed

    Al-Horani, Rami A; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K; Desai, Umesh R

    2016-01-01

    Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants. PMID:27467511

  11. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile

    PubMed Central

    Al-Horani, Rami A.; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K.

    2016-01-01

    Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants. PMID:27467511

  12. An allosteric model of the molecular interactions of excitation- contraction coupling in skeletal muscle

    PubMed Central

    1993-01-01

    A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88- 118), and analogous to one proposed by Marks and Jones for voltage- dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the

  13. Comparison of the effects of the GABAB receptor positive modulator BHF177 and the GABAB receptor agonist baclofen on anxiety-like behavior, learning, and memory in mice.

    PubMed

    Li, Xia; Risbrough, Victoria B; Cates-Gatto, Chelsea; Kaczanowska, Katarzyna; Finn, M G; Roberts, Amanda J; Markou, Athina

    2013-07-01

    γ-Aminobutyric acid B (GABAB) receptor activation is a potential therapeutic approach for the treatment of drug addiction, pain, anxiety, and depression. However, full agonists of this receptor induce side-effects, such as sedation, muscle relaxation, tolerance, and cognitive disruption. Positive allosteric modulators (PAMs) of the GABAB receptor may have similar therapeutic effects as agonists with superior side-effect profiles. The present study behaviorally characterized N-([1R,2R,4S]-bicyclo[2.2.1]hept-2-yl)-2-methyl-5-(4-[trifluoromethyl]phenyl)-4-pyrimidinamine (BHF177), a GABAB receptor PAM, in mouse models of anxiety-like behavior, learning and memory. In addition, the effects of BHF177 were compared with the agonist baclofen. Unlike the anxiolytic chlordiazepoxide, baclofen (0.5, 1.5, and 2.5 mg/kg, intraperitoneally) and BHF177 (10, 20, and 40 mg/kg, orally) had no effect on anxiety-like behavior in the elevated plus maze, light/dark box, or Vogel conflict test. Baclofen increased punished drinking in the Vogel conflict test, but this effect may be attributable to the analgesic actions of baclofen. At the highest dose tested (2.5 mg/kg), baclofen-treated mice exhibited sedation-like effects (i.e., reduced locomotor activity) across many of the tests, whereas BHF177-treated mice exhibited no sedation-like effects. BHF177 exhibited pro-convulsion properties only in mice, but not in rats, indicating that this effect may be species-specific. At doses that were not sedative or pro-convulsant, baclofen and BHF177 had no selective effects on fear memory retrieval in contextual and cued fear conditioning or spatial learning and memory in the Barnes maze. These data suggest that BHF177 has little sedative activity, no anxiolytic-like profile, and minimal impairment of learning and memory in mice.

  14. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  15. Allosteric sodium in class A GPCR signaling

    PubMed Central

    Katritch, Vsevolod; Fenalti, Gustavo; Abola, Enrique E.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2014-01-01

    Despite their functional and structural diversity, G protein-coupled receptors (GPCRs) share a common mechanism of signal transduction via conformational changes in the seven-transmembrane (7TM) helical domain. New major insights into this mechanism come from the recent crystallographic discoveries of a partially hydrated sodium ion that is specifically bound in the middle of the 7TM bundle of multiple class A GPCRs. This review discusses the remarkable structural conservation and distinct features of the Na+ pocket in this most populous GPCR class, as well as the conformational collapse of the pocket on receptor activation. New insights help to explain allosteric effects of sodium on GPCR agonist binding and activation, and sodium’s role as a potential co-factor in class A GPCR function. PMID:24767681

  16. Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species.

    PubMed

    Napoléon, Camille; Raimbault, Virginie; Claquin, Pascal

    2013-01-01

    Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method ((13)C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (Fv/Fm) values, confirming that Fv/Fm is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETR(Chl) (electron transport rate) and P(Chl) (carbon fixation rate) estimated using PAM measurements and (13)C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETR(Chl) and P(Chl) can be described by the shape and the slope of the curve (ΦC.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in ΦC.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions

  17. Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species

    PubMed Central

    Napoléon, Camille; Raimbault, Virginie; Claquin, Pascal

    2013-01-01

    Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method (13C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (Fv/Fm) values, confirming that Fv/Fm is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETRChl (electron transport rate) and PChl (carbon fixation rate) estimated using PAM measurements and 13C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETRChl and PChl can be described by the shape and the slope of the curve (ΦC.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in ΦC.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions, due to the

  18. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  19. Efficiency of various modulation types in Step Index Polymer Optical Fiber

    NASA Astrophysics Data System (ADS)

    Siuzdak, Jerzy; Stepniak, Grzegorz

    2014-11-01

    Efficiency of PAM, CAP/QAM and OFDM/DMT modulation for Step Index Polymer Optical Fiber channel is analyzed theoretically. It is shown that for the same transmitted optical power and same BER they offer similar data throughputs. However, when the light source has limited dynamics the crucial factor is the peak to average power ratio of the modulating signal. This is the greatest for OFDM/DMT and smaller for CAP and PAM. Consequently, the efficiency for PAM and CAP should be comparable whereas that of DMT - inferior. This conclusion was confirmed by experimental results.

  20. Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4.

    PubMed

    Chen, Yi-Guo; Zhang, Yong; Deng, Lin-Qiang; Chen, Hui; Zhang, Yu-Juan; Zhou, Nan-Jin; Yuan, Keng; Yu, Li-Zhi; Xiong, Zhang-Hua; Gui, Xiao-Mei; Yu, Yan-Rong; Wu, Xiao-Mu; Min, Wei-Ping

    2016-01-01

    The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1β and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-β, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia. PMID:26974438