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Sample records for a3 receptor affinity

  1. Neoceptor Concept Based on Molecular Complementarity in GPCRs: A Mutant Adenosine A3 Receptor with Selectively Enhanced Affinity for Amine-Modified Nucleosides

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Chen, Aishe; Barak, Dov; Kim, Soon-Ai; Lee, Kyeong; Link, Andreas; Van Rompaey, Philippe; van Calenbergh, Serge; Liang, Bruce T.

    2012-01-01

    Adenosine A3 receptors are of interest in the treatment of cardiac ischemia, inflammation, and neurodegenerative diseases. In an effort to create a unique receptor mutant that would be activated by tailor-made synthetic ligands, we mutated the human A3 receptor at the site of a critical His residue in TM7, previously proposed to be involved in ligand recognition through interaction with the ribose moiety. The H272E mutant receptor displayed reduced affinity for most of the uncharged A3 receptor agonists and antagonists examined. For example, the nonselective agonist 1a was 19-fold less potent at the mutant receptor than at the wild-type receptor. The introduction of an amino group on the ribose moiety of adenosine resulted in either equipotency or enhanced binding affinity at the H272E mutant relative to wild-type A3 receptors, depending on the position of the amino group. 3′-Amino-3′-deoxyadenosine proved to be 7-fold more potent at the H272E mutant receptor than at the wild-type receptor, while the corresponding 2′- and 5′-amino analogues did not display significantly enhanced affinities. An 3′-amino-N6-iodobenzyl analogue showed only a small enhancement at the mutant (Ki = 320 nM) vs wild-type receptors. The 3′-amino group was intended for a direct electrostatic interaction with the negatively charged ribose-binding region of the mutant receptor, yet molecular modeling did not support this notion. This design approach is an example of engineering the structure of mutant receptors to recognize synthetic ligands for which they are selectively matched on the basis of molecular complementarity between the mutant receptor and the ligand. We have termed such engineered receptors “neoceptors”, since the ligand recognition profile of such mutant receptors need not correspond to the profile of the parent, native receptor. PMID:11708915

  2. Synthesis and pharmacological characterization of [125I]MRS1898, a high-affinity, selective radioligand for the rat A3 adenosine receptor

    PubMed Central

    Gao, Zhan-Guo; Teng, Bao; Wu, Haitao; Joshi, Bhalchandra V.; Griffiths, Gary L.

    2008-01-01

    A known selective agonist of the A3 adenosine receptors (AR), MRS1898 [(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A3AR affinity across species, with nanomolar binding at both rat and human A3ARs. The radioiodination of MRS1898 on its N6–3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [125I]MRS1898 bound to the rat A3AR with a Kd value of 0.17 ± 0.04 nM and a Bmax value of 0.66 ± 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [125I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A3AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A3AR. PMID:18528782

  3. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  4. Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity.

    PubMed

    Gadhiya, Satishkumar; Madapa, Sudharshan; Kurtzman, Thomas; Alberts, Ian L; Ramsey, Steven; Pillarsetty, Nagavara-Kishore; Kalidindi, Teja; Harding, Wayne W

    2016-05-01

    Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds. PMID:27032890

  5. Neuroprotective effects of high affinity sigma 1 receptor selective compounds

    PubMed Central

    Luedtke, Robert R.; Perez, Evelyn; Yang, Shao-Hua; Liu, Ran; Vangveravong, Suwanna; Tu, Zhude; Mach, Robert H.; Simpkins, James W.

    2014-01-01

    We previously reported that the antipsychotic drug haloperidol, a multifunctional D2-like dopamine and sigma receptor subtype antagonist, has neuroprotective properties. In this study we further examined the association between neuroprotection and receptor antagonism by evaluating a panel of novel compounds with varying affinity at sigma and D2-like dopamine receptors. These compounds were evaluated using an in vitro cytotoxicity assay that utilizes a hippocampal-derived cell line, HT-22, in the presence or absence of varying concentrations (5 to 20 mM) of glutamate. While haloperidol was found to be a potent neuroprotective agent in this in vitro cell assay, the prototypic sigma 1 receptor agonist (+)-pentazocine was found not to be neuroprotective. Subsequently, the potency for the neuroprotection of HT-22 cells was evaluated for a) three SV series indoles which have nMolar affinity at D2-like receptors but varying affinity at sigma 1 receptor and b) two benzyl phenylacetamides sigma 1 receptor selective compounds which bind with low affinity at D2-like receptors but have nMolar affinity for the sigma 1 receptor. We observed that cytoprotection correlated with the affinity of the compounds for sigma 1 receptors. Based upon results from the HT-22 cell-based in vitro assay, two phenylacetamides, LS-127 and LS-137, were further evaluated in vivo using a transient middle cerebral artery occlusion (t-MCAO) model of stroke. At a dose of 100 µg/kg, both LS-127 and LS-137 attenuated infarct volume by approximately 50%. These studies provide further evidence that sigma 1 receptor selective compounds can provide neuroprotection in cytotoxic situations. These results also demonstrate that sigma 1 receptor selective benzyl phenylacetamides are candidate pharmacotherapeutic agents that could be used to minimize neuronal death after a stroke or head trauma. PMID:22285434

  6. Optimal T-cell receptor affinity for inducing autoimmunity.

    PubMed

    Koehli, Sabrina; Naeher, Dieter; Galati-Fournier, Virginie; Zehn, Dietmar; Palmer, Ed

    2014-12-01

    T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity. PMID:25411315

  7. Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity.

    PubMed

    Lazareno, S; Dolezal, V; Popham, A; Birdsall, N J M

    2004-01-01

    Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity. PMID:14722259

  8. Binding affinity prediction of novel estrogen receptor ligands using receptor-based 3-D QSAR methods.

    PubMed

    Sippl, Wolfgang

    2002-12-01

    We have recently reported the development of a 3-D QSAR model for estrogen receptor ligands showing a significant correlation between calculated molecular interaction fields and experimentally measured binding affinity. The ligand alignment obtained from docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection procedure, a significant and robust model was obtained (q(2)(LOO)=0.921, SDEP=0.345). To further analyze the robustness and the predictivity of the established model several recently developed estrogen receptor ligands were selected as external test set. An excellent agreement between predicted and experimental binding data was obtained indicated by an external SDEP of 0.531. Two other traditionally used prediction techniques were applied in order to check the performance of the receptor-based 3-D QSAR procedure. The interaction energies calculated on the basis of receptor-ligand complexes were correlated with experimentally observed affinities. Also ligand-based 3-D QSAR models were generated using program FlexS. The interaction energy-based model, as well as the ligand-based 3-D QSAR models yielded models with lower predictivity. The comparison with the interaction energy-based model and with the ligand-based 3-D QSAR models, respectively, indicates that the combination of receptor-based and 3-D QSAR methods is able to improve the quality of prediction. PMID:12413831

  9. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  10. RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR

    EPA Science Inventory

    RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR. T R Henry1, J S Denny2 and P K Schmieder2. USEPA, ORD, NHEERL, 1Experimental Toxicology Division and 2Mid-Continent Ecology Division, Duluth, MN, USA.
    The USEPA has been mandated to screen industria...

  11. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  12. Altered catecholamine receptor affinity in rabbit aortic intimal hyperplasia

    SciTech Connect

    O'Malley, M.K.; Cotecchia, S.; Hagen, P.O. )

    1991-08-01

    Intimal thickening is a universal response to endothelial denudation and is also thought to be a precursor of atherosclerosis. The authors have demonstrated selective supersensitivity in arterial intimal hyperplasia to norepinephrine and they now report a possible mechanism for this. Binding studies in rabbit aorta with the selective alpha 1-adrenergic radioligand 125I-HEAT demonstrated that there was no change in receptor density (20 {plus minus} 4 fmole/10(6) cells) in intact vascular smooth muscle cells at either 5 or 14 days after denudation. However, competition studies showed a 2.6-fold increase in alpha 1-adrenergic receptor affinity for norepinephrine in intimal hyperplastic tissue (P less than 0.05). This increased affinity for norepinephrine was associated with a greater increase in 32P-labeled phosphatidylinositol (148% intimal thickening versus 76% control) and phosphatidic acid (151% intimal thickening versus 56% control) following norepinephrine stimulation of free floating rings of intimal hyperplastic aorta. These data suggest that the catecholamine supersensitivity in rabbit aortic intimal hyperplasia is receptor mediated and may be linked to the phosphatidylinositol cycle.

  13. Affinity enhancement by dendritic side chains in synthetic carbohydrate receptors.

    PubMed

    Destecroix, Harry; Renney, Charles M; Mooibroek, Tiddo J; Carter, Tom S; Stewart, Patrick F N; Crump, Matthew P; Davis, Anthony P

    2015-02-01

    Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 M(-1) . Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions. PMID:25645064

  14. A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor

    PubMed Central

    VAN GALEN, PHILIP J. M.; VAN BERGEN, ANDREW H.; GALLO-RODRIGUEZ, CAROLA; MELMAN, NELI; OLAH, MARK E.; IJZERMAN, AD P.; STILES, GARY L.; JACOBSON, KENNETH A.

    2012-01-01

    SUMMARY A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at

  15. Gangliosides as high affinity receptors for tetanus neurotoxin.

    PubMed

    Chen, Chen; Fu, Zhuji; Kim, Jung-Ja P; Barbieri, Joseph T; Baldwin, Michael R

    2009-09-25

    Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a approximately 150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT. PMID:19602728

  16. Affinity of Iresine herbstii and Brugmansia arborea extracts on different cerebral receptors.

    PubMed

    Nencini, Cristina; Cavallo, Federica; Bruni, Giancarlo; Capasso, Anna; De Feo, Vincenzo; De Martino, Laura; Giorgi, Giorgio; Micheli, Lucia

    2006-05-24

    Iresine herbstii Hook. (Amaranthaceae) and Brugmansia arborea (L.) Lagerheim (Solanaceae) are used in the northern Peruvian Andes for magic-therapeutical purposes. The traditional healers use Iresine herbstii with the ritual aim to expel bad spirits from the body. Furthermore, Iresine herbstii was used in association with other plants, such as Trichocereus pachanoi Britt. et Rose, for divination, to diagnose diseases, and to take possession of another identity. Also, species of Brugmansia have been reported to be used during ritual practices for magical and curative purposes. Given the above evidence, the aim of the present study is to evaluate if the central effects of Iresine herbstii and Brugmansia arborea could be associated with interaction with SNC receptors. Two Iresine herbstii extracts (methanolic and aqueous) and one Brugmansia arborea aqueous extract were tested for in vitro affinity on 5-HT(1A), 5-HT(2A), 5-HT(2C), D1, D2, alpha(1), and alpha(2) receptors by radioligand binding assays. The biological materials for binding assay (cerebral cortex) were taken from male Sprague-Dawley rats. The extracts affinity for receptors is definite as inhibition percentage of radioligand/receptor binding and measured as the radioactivity of remaining complex radioligand/receptor. The data obtained for Iresine extracts have shown a low affinity for the 5-HT(1A) receptor and no affinity for 5-HT(2A) receptor. Otherwise the methanolic extract showed affinity for 5-HT(2C) receptor (IC(50): 34.78 microg/ml) and for D1 receptor (IC(50): 19.63 microg/ml), instead the Iresine aqueous extract displayed a lower affinity for D1 (48.3% at the maximum concentration tested) and a higher value of affinity for D2 receptors (IC(50): 32.08 microg/ml). The Brugmansia aqueous extract displayed affinity for D1 receptors (IC(50): 17.68 microg/ml), D2 receptors (IC(50): 15.95 microg/ml) and weak affinity for the serotoninergic receptors. None of the three extracts showed relevant affinity

  17. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    PubMed Central

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  18. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

    PubMed

    Kržan, Mojca; Vianello, Robert; Maršavelski, Aleksandra; Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  19. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  20. (/sup 14/C)chloroacetylcholine as an advantageous affinity label of the acetylcholine receptor

    SciTech Connect

    Bodmer, D.M.; Sin-Ren, A.C.; Waser, P.G.

    1987-01-01

    The alkylating agent (/sup 14/C)chloroacetylcholine perchlorate ((/sup 14/C) ClACh) was synthesized and used for affinity labelling of the nicotinic acetylcholine receptor from Torpedo marmorata. Solubilized and affinity-purified receptor proteins were reduced and alkylated according to the bromoacetylcholine-method. Covalent binding of (/sup 14/C) ClACh to the cholinergic receptor proved to be specific and saturable, and occurred exclusively to the alpha-subunit. Halogen substitution of acetylcholine by chlorine and insertion of a /sup 14/C-isotope instead of the widely used /sup 3/H resulted in favorable properties of the affinity label.

  1. Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

    SciTech Connect

    Borst, S.E.; Catherwood, B.D. )

    1989-04-01

    We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of (125I)EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of (125I)EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound (125I)EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, (125I)EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of (125I)EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of (125I)EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of (125I)EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in (125I)EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.

  2. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain. PMID:24269284

  3. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  4. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors.

    PubMed

    Gao, Zhan-Guo; Mamedova, Liaman K; Chen, Peiran; Jacobson, Kenneth A

    2004-11-15

    The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM). PMID:15476669

  5. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors

    PubMed Central

    Gao, Zhan-Guo; Mamedova, Liaman K.; Chen, Peiran; Jacobson, Kenneth A.

    2012-01-01

    The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM). PMID:15476669

  6. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  7. Affinity profiles of hexahydro-sila-difenidol analogues at muscarinic receptor subtypes.

    PubMed

    Lambrecht, G; Feifel, R; Wagner-Röder, M; Strohmann, C; Zilch, H; Tacke, R; Waelbroeck, M; Christophe, J; Boddeke, H; Mutschler, E

    1989-09-01

    In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1-(rabbit vas deferens), M2- (guinea-pig atria) and M3- (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 approximately M3 greater than M2. A different selectivity pattern was observed for p-fluoro-hexahydro-sila-difenidol: M3 greater than M1 greater than M2. This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the M2-receptors in guinea-pig atria (pA2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M1) or hexahydrosila-difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes. PMID:2583233

  8. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  9. Characterization of opiate receptor heterogeneity using affinity ligands and phospholipase A/sub 2/

    SciTech Connect

    Reichman, M.

    1985-01-01

    The primary aim of the dissertation was to study the heterogeneity of opiate receptors by utilizing affinity ligands, and by modification of the receptor lipid-microenvironment with phospholipase A/sub 2/ (PLA/sub 2/). The affinity ligands, 14-bromacetamidomorphine (BAM) and 14-chloroacetylnaltrexone (CAN), selectively inactivated high affinity dihydromorphine binding sites in an apparently irreversible manner (the inhibition was resistant to extensive washes of treated neural membrane homogenates). The inhibitory effect of PLA/sub 2/ (10 ng/ml) on opiate receptor subtypes was determined using (/sup 3/H)-dihydromorphine (..mu..-type agonist), (/sup 3/H)-enkephalin (delta agonist) and (/sup 3/H)-naloxone (..mu.. antagonist). PLA/sub 2/ abolished the high affinity antagonist binding site, whereas it inhibited high and low affinity agonist binding sites similarly. The results suggest that high affinity antagonist binding sites are different from high affinity agonist binding sites. Indirect binding assays demonstrated that the selectivities of ..mu..- and delta receptors are not affected significantly by PLA/sub 2/ treatment.

  10. N-Alkyl Ammonium Resorcinarene Salts as High-Affinity Tetravalent Chloride Receptors.

    PubMed

    Beyeh, N Kodiah; Pan, Fangfang; Bhowmik, Sandip; Mäkelä, Toni; Ras, Robin H A; Rissanen, Kari

    2016-01-22

    N-Alkyl ammonium resorcinarene salts (NARYs, Y=triflate, picrate, nitrate, trifluoroacetates and NARBr) as tetravalent receptors, are shown to have a strong affinity for chlorides. The high affinity for chlorides was confirmed from a multitude of exchange experiments in solution (NMR and UV/Vis), gas phase (mass spectrometry), and solid-state (X-ray crystallography). A new tetra-iodide resorcinarene salt (NARI) was isolated and fully characterized from exchange experiments in the solid-state. Competition experiments with a known monovalent bis-urea receptor (5) with strong affinity for chloride, reveals these receptors to have a much higher affinity for the first two chlorides, a similar affinity as 5 for the third chloride, and lower affinity for the fourth chloride. The receptors affinity toward chloride follows the trend K1 ≫K2 ≫K3 ≈5>K4, with Ka =5011 m(-1) for 5 in 9:1 CDCl3/[D6]DMSO. PMID:26671730

  11. Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

    SciTech Connect

    Sidhu, A.

    1988-11-29

    The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist /sup 125/I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SKandF R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.

  12. Interaction of nicotinic receptor affinity reagents with central nervous system. cap alpha. -bungarotoxin-binding entities

    SciTech Connect

    Lukas, R.J.; Bennett, E.L.

    1980-01-01

    Membrane-bound ..cap alpha..-bungarotoxin-binding entities derived from rat brain are found to interact specifically with the affinity reagents maleimidobenzyltrimethylammonium (MBTA) and bromoacetylcholine (BAC), originally designed to label nicotinic acetylcholine receptors from electroplax and skeletal muscle. Following treatment of membranes with dithiothreitol, all specific toxin binding sites are irreversibly blocked by reaction with MBTA or BAC. Affinity reagent labeling of dithiothreitol-reduced membranes is prevented (toxin binding sites are not blocked) by prior alkylaction with N-ethylmaleimide, by prior oxidation with dithiobis(2-nitrobenzoic acid), or by incubation with neurotoxin. Reversibly associating cholinergic agonists and antagonists retard the rate of affinity reagent interaction with toxin receptors. The apparent rates of affinity reagent alkylation of toxin receptors, and the influences of other sulfhydryl/disulfide reagents on affinity labeling are comparable to those observed for reaction with nicotinic acetylcholine receptors in the periphery. The results provide further evidence that central nervous system ..cap alpha..-bungarotoxin receptors share a remarkable number of biochemical properties with nicotinic receptors from the periphery.

  13. Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam.

    PubMed

    Gruen, R J; Elsworth, J D; Roth, R H

    1990-04-23

    Alterations in a low affinity form of the GABAA receptor were examined with [3H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [3H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [3H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABAA receptors are functionally linked during the perinatal period. PMID:2162709

  14. Trifluoromethoxyl Substituted Phenylethylene Diamines as High Affinity σ Receptor Ligands with Potent Anti-Cocaine Actions

    PubMed Central

    Smith, Trudy A.; Yang, Xiaowen; Wu, Huifang; Pouw, Buddy; Matsumoto, Rae R.; Coop, Andrew

    2012-01-01

    The phenylethylene diamines are a class of σ receptor ligands with excellent selectivity over other biological systems and with anti-cocaine actions that involve antagonism of σ1 receptors. In order to increase the potency of the aromatic methoxyl substituted analogues, trifluoromethoxyl groups were introduced to prevent metabolic demethylation. The para-substituted trifluoromethoxyl substituted analogues were shown to have increased σ receptor affinity and represent the most potent anti-cocaine phenylethylene diamines yet described. PMID:18461921

  15. Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

    PubMed Central

    Bolcato, Chiara; Cusan, Claudia; Pastorin, Giorgia; Cacciari, Barbara; Klotz, Karl Norbert; Morizzo, Erika

    2007-01-01

    In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach. PMID:18368532

  16. PREDICTING RETINOID RECEPTOR BINDING AFFINITY: COREPA-M APPLICATION

    EPA Science Inventory

    Retinoic acid and associated vitamin A derivatives comprise a class of endogenous hormones that activate different retinoic acid receptors RARs). Transcriptional events subsequent to this activation are key to controlling several aspects of vertebrate development. As such, identi...

  17. A(3) adenosine receptor ligands: history and perspectives.

    PubMed

    Baraldi, P G; Cacciari, B; Romagnoli, R; Merighi, S; Varani, K; Borea, P A; Spalluto, G

    2000-03-01

    Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic. PMID:10723024

  18. Functionalized Congeners of 1,4-Dihydropyridines as Antagonist Molecular Probes for A3 Adenosine Receptors

    PubMed Central

    Li, An-Hu; Chang, Louis; Ji, Xiao-duo; Melman, Neli; Jacobson, Kenneth A.

    2012-01-01

    4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure–activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 μM. PMID:10411465

  19. Effect of sodium ion on the affinity of naloxone for the kappa opioid receptor

    SciTech Connect

    Cheney, B.V.; Lahti, R.A.

    1987-03-16

    Several investigators have observed that sodium ion enhances the binding of naloxone to opioid receptors. This effect has generally been attributed to allosteric modulation of the state of the mu receptor. However, a recent claim has been made that the enhancement does not involve a change in the mu receptor, but instead occurs because naloxone becomes a more kappa-specific drug when sodium ion is present in high concentration. Since the claim was not based on experimental evidence from binding studies involving known high-affinity kappa ligands, the authors have investigated the competition of naloxone for the kappa site using (/sup 3/H)U-69593 as the marker for receptor binding. Assays were carried out in the presence and absence of 100 mM NaCl. The results of the study indicate that sodium ion does not increase the affinity of naloxone or U-69593 for the kappa receptor. 9 references, 1 figure.

  20. Synthesis and estrogen receptor affinity of a 4-hydroxytamoxifen-labeled ligand for diagnostic imaging.

    PubMed

    Lashley, Matthew R; Niedzinski, Edmund J; Rogers, Jane M; Denison, Michael S; Nantz, Michael H

    2002-12-01

    A 10-step synthesis of a novel 4-hydroxytamoxifen-DTPA ligand (HOTam-DTPA) is reported. Tamoxifen and its primary metabolite 4-hydroxytamoxifen are common estrogen receptor ligands. Consequently, tamoxifen has found utility as the targeting component of various diagnostic agents for selective imaging of estrogen receptor-rich tissue, specifically breast cancer. An L-aspartic acid-derived DTPA analogue was attached to the ethyl side chain of 4-hydroxy-tamoxifen using N,N'-dimethylethylenediamine as a hydrophilic linker. A competitve estrogen receptor binding assay using [3H]-17beta-estradiol was performed to determine the effect of the ethyl side chain modification on estrogen receptor affinity. The results show that while the relative affinity of HOTam-DTPA for the estrogen receptor is approximately 10-fold lower than that of tamoxifen, it still remains a potent ligand at relatively low concentrations. PMID:12413861

  1. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  2. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  3. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

  4. Pyran Template Approach to the Design of Novel A3 Adenosine Receptor Antagonists

    PubMed Central

    Li, An-Hu; Ji, Xiao-duo; Kim, Hak Sung; Melman, Neli; Jacobson, Kenneth A.

    2016-01-01

    Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I–III Regulatory, Quality, ManufacturingPostmarketing Phase IV A3 adenosine receptor antagonists have potential as anti-inflammatory, anti-asthmatic, and anti-ischemic agents. We previously reported the preparation of chemical libraries of 1,4-dihydropyridine (DHP) and pyridine derivatives and identification of members having high affinity at A3 adenosine receptors. These derivatives were synthesized through standard three-component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.e., the central ring served as a molecular scaffold for structurally diverse substituents. We extended this template approach from the DHP series to chemically stable pyran derivatives, in which the ring NH is replaced by O and which is similarly derived from a stepwise reaction of three components. Since the orientation of substituent groups may be conformationally similar to the 1,4-DHPs, a direct comparison between the structure activity relationships of key derivatives in binding to adenosine receptors was carried out. Affinity at human A3 receptors expressed in CHO cells was determined vs. binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methyl-carbamoyladenosine). There was no potency-enhancing effect, as was observed for DHPs, of 4-styryl, 4-phenylethynyl, or 6-phenyl substitutions. The most potent ligands in this group in binding to human A3 receptors were 6-methyl and 6-phenyl analogs, 3a (MRS 1704) and 4a (MRS 1705), respectively, of 3,5-diethyl 2-methyl-4-phenyl-4H-pyran-3,5-dicarboxylate, which had Ki values of 381 and 583 nM, respectively. These two derivatives were selective for human A3 receptors vs. rat brain A1 receptors by 57-fold and 24-fold, respectively. These derivatives were inactive in binding at rat brain A

  5. CORAL: prediction of binding affinity and efficacy of thyroid hormone receptor ligands.

    PubMed

    Toropova, A P; Toropov, A A; Benfenati, E

    2015-08-28

    Quantitative structure - activity relationships (QSARs) for binding affinity of thyroid hormone receptors based on attributes of molecular structure extracted from simplified molecular input-line entry systems (SMILES) are established using the CORAL software (http://www.insilico.eu/coral). The half maximal inhibitory concentration (IC50) is used as the measure of the binding affinity of thyroid hormone receptors. Molecular features which are statistically reliable promoters of increase and decrease for IC50 are suggested. The examples of modifications of molecular structure which lead to the increase or to the decrease of the endpoint are represented. PMID:26188619

  6. Molecular docking approaches in identification of High affinity inhibitors of Human SMO receptor

    PubMed Central

    Akare, Uday Raj; Bandaru, Srinivas; Shaheen, Uzma; Singh, Pramod Kumar; Tiwari, Geet; Singare, Paramanand; Nayarisseri, Anuraj; Banerjee, Tushar

    2014-01-01

    Inappropriate activation of the Hh signaling pathway has been implicated in the development of several types of cancers including prostate, lung, pancreas, breast, brain and skin. Present study identified the binding affinities of eight established inhibitors viz., Cyclopamine, Saridegib, Itraconazole, LDE-225, TAK-441, BMS-833923 (XL139), PF-04449913 and Vismodegib targeting SMO receptor - a candidate protein involved in hedgehog pathway and sought to identify the best amongst the established inhibitors through by molecular docking. Exelxis® BMS 833923 (XL 139) demonstrated superior binding affinity aided by MolDock scoring docking algorithm. Further BMS 833923 (XL 139) was evaluated for pharmacophoric features which revealed appreciable ligand receptor interactions. PMID:25670876

  7. Metabolic mapping of A3 adenosine receptor agonist MRS5980.

    PubMed

    Fang, Zhong-Ze; Tosh, Dilip K; Tanaka, Naoki; Wang, Haina; Krausz, Kristopher W; O'Connor, Robert; Jacobson, Kenneth A; Gonzalez, Frank J

    2015-09-15

    (1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is an A3AR selective agonist containing multiple receptor affinity- and selectivity-enhancing modifications and a therapeutic candidate drug for many inflammatory diseases. Metabolism-related poor pharmacokinetic behavior and toxicities are a major reason for drug R&D failure. Metabolomics with UPLC-MS was employed to profile the metabolism of MRS5980 and MRS5980-induced disruption of endogenous compounds. Recombinant drug-metabolizing enzymes screening experiment were used to determine the enzymes involved in MRS5980 metabolism. Analysis of lipid metabolism-related genes was performed to investigate the reason for MRS5980-induced lipid metabolic disorders. Unsupervised principal components analysis separated the control and MRS5980 treatment groups in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation of feces and urine were oxidized MRS5980, glutathione (GSH) conjugates and cysteine conjugate (degradation product of the GSH conjugates) of MRS5980. The major ions contributing to the group separation of liver samples were phosphatidylcholines. In vitro incubation experiments showed the involvement of CYP3A enzymes in the oxidative metabolism of MRS5980 and direct GSH reactivity of MRS5980. The electrophilic attack by MRS5980 is a minor pathway and did not alter GSH levels in liver or liver histology, and thus may be of minor clinical consequence. Gene expression analysis further showed decreased expression of PC biosynthetic genes choline kinase a and b, which further accelerated conversion of lysophosphatidylcholine to phosphatidylcholines through increasing the expression of lysophosphatidylcholine acyltransferase 3. These data will be useful to guide rational design of drugs targeting A3AR, considering efficacy, metabolic elimination, and

  8. GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells.

    PubMed

    Meier, E; Drejer, J; Schousboe, A

    1984-12-01

    The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors. PMID:6149269

  9. Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.

    PubMed Central

    Graham, R M; Hess, H J; Homcy, C J

    1982-01-01

    The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization. PMID:6285370

  10. Computational design of the affinity and specificity of a therapeutic T cell receptor.

    PubMed

    Pierce, Brian G; Hellman, Lance M; Hossain, Moushumi; Singh, Nishant K; Vander Kooi, Craig W; Weng, Zhiping; Baker, Brian M

    2014-02-01

    T cell receptors (TCRs) are key to antigen-specific immunity and are increasingly being explored as therapeutics, most visibly in cancer immunotherapy. As TCRs typically possess only low-to-moderate affinity for their peptide/MHC (pMHC) ligands, there is a recognized need to develop affinity-enhanced TCR variants. Previous in vitro engineering efforts have yielded remarkable improvements in TCR affinity, yet concerns exist about the maintenance of peptide specificity and the biological impacts of ultra-high affinity. As opposed to in vitro engineering, computational design can directly address these issues, in theory permitting the rational control of peptide specificity together with relatively controlled increments in affinity. Here we explored the efficacy of computational design with the clinically relevant TCR DMF5, which recognizes nonameric and decameric epitopes from the melanoma-associated Melan-A/MART-1 protein presented by the class I MHC HLA-A2. We tested multiple mutations selected by flexible and rigid modeling protocols, assessed impacts on affinity and specificity, and utilized the data to examine and improve algorithmic performance. We identified multiple mutations that improved binding affinity, and characterized the structure, affinity, and binding kinetics of a previously reported double mutant that exhibits an impressive 400-fold affinity improvement for the decameric pMHC ligand without detectable binding to non-cognate ligands. The structure of this high affinity mutant indicated very little conformational consequences and emphasized the high fidelity of our modeling procedure. Overall, our work showcases the capability of computational design to generate TCRs with improved pMHC affinities while explicitly accounting for peptide specificity, as well as its potential for generating TCRs with customized antigen targeting capabilities. PMID:24550723

  11. Impact of D2 Receptor Internalization on Binding Affinity of Neuroimaging Radiotracers

    PubMed Central

    Guo, Ningning; Guo, Wen; Kralikova, Michaela; Jiang, Man; Schieren, Ira; Narendran, Raj; Slifstein, Mark; Abi-Dargham, Anissa; Laruelle, Marc; Javitch, Jonathan A; Rayport, Stephen

    2010-01-01

    Synaptic dopamine (DA) levels seem to affect the in vivo binding of many D2 receptor radioligands. Thus, release of endogenous DA induced by the administration of amphetamine decreases ligand binding, whereas DA depletion increases binding. This is generally thought to be due to competition between endogenous DA and the radioligands for D2 receptors. However, the temporal discrepancy between amphetamine-induced increases in DA as measured by microdialysis, which last on the order of 2 h, and the prolonged decrease in ligand binding, which lasts up to a day, has suggested that agonist-induced D2 receptor internalization may contribute to the sustained decrease in D2 receptor-binding potential seen following a DA surge. To test this hypothesis, we developed an in vitro system showing robust agonist-induced D2 receptor internalization following treatment with the agonist quinpirole. Human embryonic kidney 293 (HEK293) cells were stably co-transfected with human D2 receptor, G-protein-coupled receptor kinase 2 and arrestin 3. Agonist-induced D2 receptor internalization was demonstrated by fluorescence microscopy, flow cytometry, and radioligand competition binding. The binding of seven D2 antagonists and four agonists to the surface and internalized receptors was measured in intact cells. All the imaging ligands bound with high affinity to both surface and internalized D2 receptors. Affinity of most of the ligands to internalized receptors was modestly lower, indicating that internalization would reduce the binding potential measured in imaging studies carried out with these ligands. However, between-ligand differences in the magnitude of the internalization-associated affinity shift only partly accounted for the data obtained in neuroimaging experiments, suggesting the involvement of mechanisms beyond competition and internalization. PMID:19956086

  12. Rational design of high affinity tachykinin NK1 receptor antagonists.

    PubMed

    Boyle, S; Guard, S; Higginbottom, M; Horwell, D C; Howson, W; McKnight, A T; Martin, K; Pritchard, M C; O'Toole, J; Raphy, J

    1994-05-01

    The rational design of a non-peptide tachykinin NK1 receptor antagonist, [(2-benzofuran)-CH2OCO]-(R)-alpha-MeTrp-(S)-NHCH(CH3)P h (28, PD 154075) is described. Compound 28 has a Ki = 9 and 0.35 nM for the NK1 receptor binding site in guinea-pig cerebral cortex membranes and human IM9, cells respectively (using [125I] Bolton-Hunter-SP as the radioligand). It is a potent antagonist in vitro where it antagonises the contractions mediated by SPOMe in the guinea-pig ileum (KB = 0.3 nM). Compound 28 is active in vivo in the guinea-pig plasma extravasation model, where it is able to block the SPOMe-induced protein plasma extravasation (monitored by Evans Blue) in the bladder with an ID50 of 0.02 mg kg-1 iv. PMID:7922147

  13. Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

    PubMed

    Hill, R J; Oleynek, J J; Hoth, C F; Kiron, M A; Weng, W; Wester, R T; Tracey, W R; Knight, D R; Buchholz, R A; Kennedy, S P

    1997-01-01

    The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in

  14. Role of T Cell Receptor Affinity in the Efficacy and Specificity of Adoptive T Cell Therapies

    PubMed Central

    Stone, Jennifer D.; Kranz, David M.

    2013-01-01

    Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional αβ T cell receptor (TCR) against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR) consisting of a single-chain antibody as an Fv fragment linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the αβ TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher-affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher-affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly. PMID:23970885

  15. Three amino acids in the D2 dopamine receptor regulate selective ligand function and affinity

    PubMed Central

    Cummings, David F.; Ericksen, Spencer S.; Schetz, John A.

    2016-01-01

    The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype-selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120-fold) for the D4 subtype of dopamine receptor were tested on wild-type and mutant D2 receptors. Five of the selective ligands were observed to have 21-fold to 293-fold increases in D2 receptor affinity when three non-conserved amino acids in TM2 and TM3 were mutated to the corresponding D4 amino acids. The two ligands with the greatest improvement in affinity for the D2 mutant receptor [i.e., 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (L-750,667) and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild-type receptor, concentrations of L-750,667 or RBI-257 that produced large reductions in the potency of quinpirole’s functional response in the mutant did not significantly reduce quinpirole’s functional response in the wild-type D2 receptor. In contrast to RBI-257 which is an antagonist at all receptors, L-750,667 is a partial agonist at the wild-type D2 but an antagonist at both the mutant D2 and wild-type D4 receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D2 dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non-conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype

  16. IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

    SciTech Connect

    Taketazu, F.; Chiba, S.; Shibuya, K.; Kuwaki, T.; Tsumura, H.; Miyazono, K.; Miyagawa, K.; Takaku, F. )

    1991-02-01

    The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF binding to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.

  17. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  18. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  19. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    NASA Astrophysics Data System (ADS)

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  20. The A3 adenosine receptor (A3AR): therapeutic target and predictive biological marker in rheumatoid arthritis.

    PubMed

    Fishman, Pnina; Cohen, Shira

    2016-09-01

    The Gi protein-associated A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells, and this high expression is also reflected in the peripheral blood mononuclear cells of patients with autoimmune inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn's disease. CF101, a selective agonist with high affinity to the A3AR, is known to induce robust anti-inflammatory effect in experimental animal models of adjuvant-, collagen-, and tropomyosin-induced arthritis. The effect is mediated via a definitive molecular mechanism entailing deregulation of the nuclear factor-κB (NF-κB) and the Wnt signal transduction pathways resulting in apoptosis of inflammatory cells. CF101 was found to be safe and well tolerated in all preclinical, phase I, and phase II human clinical studies. In two phase II clinical studies where CF101 was administered to rheumatoid arthritis (RA) patients as a stand-alone drug, a significant anti-rheumatic effect and a direct significant correlation were found between receptor expression at baseline and patients' response to the drug, suggesting that A3AR may be utilized as a predictive biomarker. The A3AR is a promising therapeutic target in rheumatoid arthritis and can be used also as a biological marker to predict patients' response to CF101. This is a unique type of a personalized medicine approach which may pave the way for a safe and efficacious treatment for this patient population. PMID:26886128

  1. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases.

    PubMed

    Silveira, C L; Eldefrawi, A T; Eldefrawi, M E

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic

  2. Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives.

    PubMed

    Rios, Pablo; Carter, Tom S; Mooibroek, Tiddo J; Crump, Matthew P; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T; Boons, Geert-Jan; Davis, Anthony P

    2016-03-01

    The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc-β-OMe with Ka ≈20,000 m(-1), whereas the other one binds an O-GlcNAcylated peptide with Ka ≈70,000 m(-1). These values substantially exceed those usually measured for GlcNAc-binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl-peptide complex can be explained by extra-cavity interactions, raising the possibility of a family of complementary receptors for O-GlcNAc in different contexts. PMID:26822115

  3. Prediction of receptor properties and binding affinity of ligands to benzodiazepine/GABAA receptors using artificial neural networks.

    PubMed

    Maddalena, D J; Johnston, G A

    1995-02-17

    To date the use of artificial neural networks (ANNs) in quantitative structure-activity relationship (QSAR) studies has been primarily concerned in comparing the predictive accuracy of the technique using known data sets where the data set parameters had been preselected and optimized for use with other statistical methods. Little effort has been directed at optimizing the input parameters for use with ANNs or exploring other potential strengths of ANNs. In this study, back-propagation ANNs and multilinear regression (MLR) were used to examine the QSAR between substituent constants and random noise at six positions on 57 1,4-benzodiazepin-2-ones (1,4-BZs) and their binding affinities (log IC50) for benzodiazepine GABAA receptor preparations. By using selective pruning and cross-validation techniques, it was found possible to use ANNs to indicate an optimum set of 10 input parameters from a choice of 48 which were then used to train back-propagation ANNs that best predicted the receptor binding affinity with a high correlation between known and predicted data sets. Using the optimum set of input parameters, three-layer ANNs performed no better than the two-layer ANNs which gave marginally better results than MLR. Using the trained ANNs to examine the individual parameters showed that increases in the lipophilicity and F polar value at position 7, F polar value at position 2', and dipole at position 1 on the molecule all enhanced receptor binding affinity of 1,4-BZ ligands. Increases in molar refractivity and resonance parameters at position 1, molar refractivity at positions 6' and 2', Hammet meta constant at position 3', and Hammet para constant at position 8 on the molecule all caused decreases in receptor binding affinity. By considering the optimal ANNs as pharmacophore models representing the internal physicochemical structure of the receptor site, it was found that they could be used to critically examine the properties of the receptor site. PMID:7861419

  4. Importin {beta}-type nuclear transport receptors have distinct binding affinities for Ran-GTP

    SciTech Connect

    Hahn, Silvia; Schlenstedt, Gabriel

    2011-03-18

    Highlights: {yields} Determination of binding properties of nuclear transport receptor/Ran-GTP complexes. {yields} Biosensor measurements provide constants for dissociation, on-rates, and off-rates. {yields} The affinity of receptors for Ran-GTP is widely divergent. {yields} Dissociation constants differ for three orders of magnitude. {yields} The cellular concentration of yeast Ran is not limiting. -- Abstract: Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin {beta} family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of {beta}-receptors and of other Ran-binding proteins was determined. We found that the number of {beta}-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

  5. Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor.

    PubMed

    Nakamura, Taichi; Ramos-Álvarez, Irene; Iordanskaia, Tatiana; Moreno, Paola; Mantey, Samuel A; Jensen, R T

    2016-09-01

    Bombesin-receptor-subtype-3 (BB3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB2 receptor)/neuromedin-B receptors (BB1 receptor). There is increased interest in BB3 receptor because studies primarily from knockout-mice suggest it plays roles in energy/glucose metabolism, insulin-secretion, as well as motility and tumor-growth. Investigations into its roles in physiological/pathophysiological processes are limited because of lack of selective ligands. Recently, a selective, peptide-antagonist, Bantag-1, was described. However, because BB3 receptor has low-affinity for all natural, Bn-related peptides, little is known of the molecular basis of its high-affinity/selectivity. This was systematically investigated in this study for Bantag-1 using a chimeric-approach making both Bantag-1 loss-/gain-of-affinity-chimeras, by exchanging extracellular (EC) domains of BB3/BB2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB3 receptor over BB2/BB1 receptors and substitution of the first EC-domain (EC1) in loss-/gain-of affinity-chimeras greatly affected affinity. Mutagenesis of each amino acid difference in EC1 between BB3 receptor/BB2 receptor showed replacement of His(107) in BB3 receptor by Lys(107) (H107K-BB3 receptor-mutant) from BB2 receptor, decreased affinity 60-fold, and three replacements [H107K, E11D, G112R] decreased affinity 500-fold. Mutagenesis in EC1's surrounding transmembrane-regions (TMs) demonstrated TM2 differences were not important, but R127Q in TM3 alone decreased affinity 400-fold. Additional mutants in EC1/TM3 explored the molecular basis for these changes demonstrated in EC1, particularly important is the presence of aromatic-interactions by His(107), rather than hydrogen-bonding or charge-charge interactions, for determining

  6. Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

    PubMed

    Rodrigues de Miranda, J F; Scheres, H M; Salden, H J; Beld, A J; Klaassen, A B; Kuijpers, W

    1985-07-31

    Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites. PMID:3840092

  7. Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

    PubMed

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dottore, Mara; McClure, Barbara J; Dhagat, Urmi; Taing, Houng; Gorman, Michael A; King-Scott, Jack; Lopez, Angel F; Parker, Michael W

    2016-08-01

    The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while βc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal. PMID:27396825

  8. Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity.

    PubMed

    Piekielna, Justyna; Kluczyk, Alicja; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tomböly, Csaba; Łapiński, Krzysztof; Janecki, Tomasz; Janecka, Anna

    2015-06-01

    The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied. PMID:25948019

  9. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  10. 2-Triazole-Substituted Adenosines: A New Class of Selective A3 Adenosine Receptor Agonists, Partial Agonists, and Antagonists

    PubMed Central

    Cosyn, Liesbet; Palaniappan, Krishnan K.; Kim, Soo-Kyung; Duong, Heng T.; Gao, Zhan-Guo; Jacobson, Kenneth A.; Van Calenbergh, Serge

    2016-01-01

    “Click chemistry” was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1–14). Binding affinity at the human A1, A2A, and A3ARs (adenosine receptors) and relative efficacy at the A3AR were determined. Some triazol-1-yl analogues showed A3AR affinity in the low nanomolar range, a high ratio of A3/A2A selectivity, and a moderate-to-high A3/A1 ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A3AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A3AR efficacy. Thus, several 5′-OH derivatives appeared to be selective A3AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A1AR and displaying a characteristic docking mode in an A3AR model. The corresponding 5′-ethyluronamide analogues generally showed increased A3AR affinity and behaved as full agonists, i.e., 17, with 910-fold A3/A1 selectivity. Thus, N6-substituted 2-(1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A3AR antagonists, partial agonists, and agonists. PMID:17149867

  11. Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay.

    PubMed

    Martínez-Pinilla, Eva; Rabal, Obdulia; Reyes-Resina, Irene; Zamarbide, Marta; Navarro, Gemma; Sánchez-Arias, Juan A; de Miguel, Irene; Lanciego, José L; Oyarzabal, Julen; Franco, Rafael

    2016-09-01

    Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may

  12. Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system

    SciTech Connect

    Escandon, E.; Chao, M.V. )

    1990-12-01

    In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with {sup 125}I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system.

  13. Differentiation of extrastriatal dopamine D2 receptor density and affinity in the human brain using PET.

    PubMed

    Olsson, Hans; Halldin, Christer; Farde, Lars

    2004-06-01

    Dopaminergic neurotransmission in extrastriatal regions may play a crucial role in the pathophysiology and treatment of neuropsychiatric disorders. The high-affinity radioligands [(11)C]FLB 457, [(123)I]epidepride, and [(18)F]fallypride are now used in clinical studies to measure these low-density receptor populations in vivo. However, a single determination of the regional binding potential (BP) does not differentiate receptor density (B(max)) from the apparent affinity (K(D)). In this positron emission tomography (PET) study, we measured extrastriatal dopamine D2 receptor density (B(max)) and apparent affinity (K(D)) in 10 healthy subjects using an in vivo saturation approach. Each subject participated in two to three PET measurements with different specific radioactivity of [(11)C]FLB 457. The commonly used simplified reference tissue model (SRTM) was used in a comparison of BP values with the B(max) values obtained from the saturation analysis. The calculated regional receptor density values were of the same magnitude (0.33-1.68 nM) and showed the same rank order as reported from postmortem studies, that is, in descending order thalamus, lateral temporal cortex, anterior cinguli, and frontal cortex. The affinity ranged from 0.27 to 0.43 nM, that is, approximately 10-20 times the value found in vitro (20 pM). The area under the cerebellar time activity curve (TAC) was slightly lower (11 +/- 8%, mean +/- SD, P = 0.004, n = 10) after injection of low as compared with high specific radioactivity, indicating sensitivity to the minute density of dopamine D2 receptors in the this region. The results of the present study support that dopamine D2 receptor density and affinity can be differentiated in low-density regions using a saturation approach. There was a significant (P < 0.001) correlation between the binding potential calculated with SRTM and the receptor density (B(max)), which supports the use of BP in clinical studies where differentiation of B(max) and K

  14. N-substituted derivatives of 4-piperidinyl benzilate: Affinities for brain muscarinic acetylcholine receptors

    SciTech Connect

    Tejani-Butt, S.M.; Luthin, G.R.; Wolfe, B.B.; Brunswick, D.J. )

    1990-01-01

    N-Substituted derivatives of 4-piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4-Piperidinyl benzilate exhibited a K{sub i} value of 2.0nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The K{sub i} values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p-fluorobenzyl, 3.0; phenethyl, 8.0; p-nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions as well or better than straight chain or branched alkyl substitutions.

  15. Progress toward a high-affinity allosteric enhancer at muscarinic M1 receptors.

    PubMed

    Lazareno, Sebastian; Popham, Angela; Birdsall, Nigel J M

    2003-01-01

    Loss of forebrain acetylcholine is an early neurochemical lesion in Alzheimer's disease (AD). As muscarinic acetylcholine receptors are involved in memory and cognition, a muscarinic agonist could therefore provide a "replacement therapy" in this disease. However, muscarinic receptors occur throughout the CNS and the periphery. A selective locus of action of a muscarinic agonist is therefore crucial in order to avoid intolerable side effects. The five subtypes of muscarinic receptors, M1-M5, have distinct regional distributions with M2 and M3 receptors mediating most of the peripheral effects. M1 receptors are the major receptor subtype in the cortex and hippocampus-the two brain regions most associated with memory and cognition. This localization has led to a, so far unsuccessful, search for a truly M1-selective muscarinic agonist. However, acetylcholinesterase inhibitors, such as donepezil (Aricept), which potentiate cholinergic neurotransmission, do have a therapeutic role in the management of AD and so the M1 receptor remains a viable therapeutic target. Our approach is to develop muscarinic allosteric enhancers-compounds that bind to the receptor at an "allosteric" site, which is distinct from the "primary" site to which ACh binds, and which enhance ACh affinity (or efficacy). Having discovered that a commercially available compound, WIN 62577, is an allosteric enhancer with micromolar potency at M3 receptors, we report here some results of a chemical synthesis project to develop this hit. Modification of WIN 62577 has led to compounds with over 1000-fold increased affinity but, so far, none of these extremely potent compounds are allosteric enhancers. PMID:14501021

  16. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

    Yan, Heng; Zhang, Enshui; Feng, Chang; Zhao, Xin

    2016-10-01

    Neuropathy is the most common diabetic complication. Although the A1 and A2A adenosine receptors are important pharmacological targets in alleviating diabetic neuropathy, the role of the A3 adenosine receptor remains unknown. Because the A3 adenosine receptor regulates pain induced by chronic constriction injury or chemotherapy, its stimulation might also attenuate diabetic neuropathy. This study examines the effects of systemic treatment with the A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA) on diabetic neuropathy and explores the putative mechanisms underlying its pharmacological effects. We show that IB-MECA alleviated mechanical hyperalgesia and thermal hypoalgesia in mice 2 weeks but not 4 weeks after streptozocin (STZ) treatment. Furthermore, IB-MECA prevented the reduction in sciatic motor nerve conduction velocity and sensory nerve conduction velocity in diabetic mice 2 weeks but not 4 weeks after STZ treatment. Similarly, IB-MECA inhibited the activation of nuclear factor-κB and decreased the generation of tumor necrosis factor-α in the spinal cord of mice 2 weeks but not 4 weeks after STZ treatment. These phenomena were associated with reduction of A3 adenosine receptor expression in the spinal cord after long-term diabetes. Our results suggest that the A3 adenosine receptor plays a critical role in regulating diabetic neuropathy and that reduction in A3 adenosine receptor expression/function might contribute to the progression of diabetic neuropathy. © 2016 Wiley Periodicals, Inc. PMID:27319979

  17. Modal affinities of endplate acetylcholine receptors caused by loop C mutations

    PubMed Central

    Vij, Ridhima; Purohit, Prasad

    2015-01-01

    The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity. PMID:26503719

  18. Bodilisant—A Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand

    PubMed Central

    2012-01-01

    A piperidine-based lead structure for the human histamine H3 receptor (hH3R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH3R ligand with affinity in the nanomolar concentration range (Ki hH3R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH3R preference. Bodilisant was used for visualization of hH3R in hH3R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH3R. PMID:24900647

  19. Rational development of high-affinity T-cell receptor-like antibodies.

    PubMed

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A; Cerundolo, Vincenzo; Jones, E Yvonne; Renner, Christoph

    2009-04-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  20. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  1. Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.

    PubMed Central

    Burton, J; Goldman, C K; Rao, P; Moos, M; Waldmann, T A

    1990-01-01

    Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction. Images PMID:1976256

  2. Pharmacological and Therapeutic Effects of A3 Adenosine Receptor (A3AR) Agonists

    PubMed Central

    Fishman, Pnina; Bar-Yehuda, Sara; Liang, Bruce T.; Jacobson, Kenneth A.

    2011-01-01

    The Gi-coupled A3 adenosine receptor (A3AR) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A3AR as a potential therapeutic target. Highly selective A3AR agonists have been synthesized and molecular recognition in the binding site has been characterized. The present review summarizes preclinical and clinical human studies demonstrating that A3AR agonists induce specific anti-inflammatory and anticancer effects via a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. Currently, A3AR agonists are being developed for the treatment of inflammatory diseases including rheumatoid arthritis and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis. PMID:22033198

  3. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    SciTech Connect

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

    2011-02-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17{beta} to the four rainbow trout ER isoforms with that of three known environmental estrogens 17{alpha}-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ER{alpha} subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17{beta}, bisphenol A binds less strongly to all four receptors, 17{alpha}-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the {alpha} subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  4. The A3 adenosine receptor: history and perspectives.

    PubMed

    Borea, Pier Andrea; Varani, Katia; Vincenzi, Fabrizio; Baraldi, Pier Giovanni; Tabrizi, Mojgan Aghazadeh; Merighi, Stefania; Gessi, Stefania

    2015-01-01

    By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health. PMID:25387804

  5. Development of novel cellular model for affinity studies of histamine H(4) receptor ligands.

    PubMed

    Karcz, Tadeusz; Kieć-Kononowicz, Katarzyna

    2013-01-01

    The G protein-coupled histamine H4 receptor (H4R) is the last member of histamine receptors family discovered so far. Its expression pattern, together with postulated involvement in a wide variety of immunological and inflammatory processes make histamine H4 receptor an interesting target for drug development. Potential H4R ligands may provide an innovative therapies for different immuno-based diseases, including allergy, asthma, pruritus associated with allergy or autoimmune skin conditions, rheumatoid arthritis and pain. However, none of successfully developed selective and potent histamine H4 receptor ligands have been introduced to the market up to date. For that reason there is still a strong demand for pharmacological models to be used in studies on potent H4R ligands. In current work we present the development of novel mammalian cell line, stably expressing human histamine H4 receptor, with use of retroviral transduction approach. Obtained cell line was pharmacologically characterized in radioligand binding studies and its utility for affinity testing of potent receptor ligands was confirmed in comparative studies with the use of relevant insect cells expression model. Obtained results allow for statement that developed cellular model may be successfully employed in search for new compounds active at histamine H4 receptor. PMID:24432340

  6. Sensitivity of binding of high-affinity dopamine receptor radioligands to increased synaptic dopamine.

    PubMed

    Gatley, S J; Gifford, A N; Carroll, F I; Volkow, N D

    2000-12-15

    PET and SPECT studies have documented that D2 radioligands of moderate affinity, but not radioligands of high affinity, are sensitive to pharmacological challenges that alter synaptic dopamine levels. The objective of this work was to determine whether the brain kinetics of high-affinity radioligands for dopamine D1 ([(3)H]SCH 23390) and D2 ([(123)I]epidepride) receptors were altered by a prolonged elevation of synaptic dopamine induced by the potent cocaine analog RTI-55. Mice were injected intravenously with radioligands either 30 min after or 4 h before intraperitoneal administration of RTI-55 (2 mg/kg). In separate experiments, the pharmacological effects of RTI-55 were assessed biochemically by measuring uptake of dopamine in synaptosomes prepared from RTI-treated mice and behaviorally by monitoring locomotor activity. Consistent with the expected elevation of synaptic dopamine, RTI-55 induced a long-lasting decrement in dopamine uptake measured ex vivo, and a prolonged increase in locomotor activity. RTI-55 injected prior to the radioligands induced a significant (P < 0.05) increase in striatal concentration of [(123)I]epidepride at 15 min, relative to saline-treated controls, but there were no differences between the two groups at later time-points. For [(3)H]SCH 23390, both initial striatal uptake and subsequent clearance were slightly increased by preadministration of RTI-55. Administration of RTI-55 4 h after the radioligands (i.e., when it was presumed that a state of near equilibrium binding of the radioligands had been reached), was associated with a significant reduction of striatal radioactivity for both radiotracers. Our results are consistent with increased competition between dopamine and radioligand for binding to both D1 and D2 receptors after treatment with RTI-55. We suggest that the magnitude of the competition is reduced by failure of the receptor binding of high-affinity radioligands to rapidly attain equilibrium. PMID:11044896

  7. Progress in the discovery of selective, high affinity A(2B) adenosine receptor antagonists as clinical candidates.

    PubMed

    Kalla, Rao V; Zablocki, Jeff

    2009-03-01

    The selective, high affinity A(2B) adenosine receptor (AdoR) antagonists that were synthesized by several research groups should aid in determining the role of the A(2B) AdoR in inflammatory diseases like asthma or rheumatoid arthritis (RA) and angiogenic diseases like diabetic retinopathy or cancer. CV Therapeutics scientists discovered the selective, high affinity A(2B) AdoR antagonist 10, a 8-(4-pyrazolyl)-xanthine derivative [CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM] that has favorable pharmacokinetic (PK) properties (t (1/2) = 4 h and F > 35% rat). Compound 10 demonstrated functional antagonism at the A(2B) AdoR (K(B) = 6 nM) and efficacy in a mouse model of asthma. In two phase 1 clinical trials, CVT-6883 was found to be safe, well tolerated, and suitable for once daily dosing. A second compound 20, 8-(5-pyrazolyl)-xanthine, has been nominated for development from Baraldi's group in conjunction with King Pharmaceuticals that has favorable A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 1,000; and K(i)(hA(3)) > 1,000 nM], and it has been demonstrated to be a functional antagonist. A third compound 32, a 2-aminopyrimidine, from the Almirall group has high A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM], and 32 has been moved into preclinical safety testing. Since three highly selective, high affinity A(2B) AdoR antagonists have been nominated for development with 10 (CVT-6883) being the furthest along in the development process, the role of the A(2B) AdoR in various disease states will soon be established. PMID:18568423

  8. Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1

    PubMed Central

    Lebbink, Robert Jan; de Ruiter, Talitha; Adelmeijer, Jelle; Brenkman, Arjan B.; van Helvoort, Joop M.; Koch, Manuel; Farndale, Richard W.; Lisman, Ton; Sonnenberg, Arnoud; Lenting, Peter J.; Meyaard, Linde

    2006-01-01

    Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell–cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. PMID:16754721

  9. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    SciTech Connect

    Feng Xian Lu; Aiyar, N.; Chaiken, I. )

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  10. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-04-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

  11. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    PubMed Central

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  12. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors.

    PubMed

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  13. Network-of-queues approach to B-cell-receptor affinity discrimination

    NASA Astrophysics Data System (ADS)

    Felizzi, Federico; Comoglio, Federico

    2012-06-01

    The immune system is one of the most complex signal processing machineries in biology. The adaptive immune system, consisting of B and T lymphocytes, is activated in response to a large spectrum of pathogen antigens. B cells recognize and bind the antigen through B-cell receptors (BCRs) and this is fundamental for B-cell activation. However, the system response is dependent on BCR-antigen affinity values that span several orders of magnitude. Moreover, the ability of the BCR to discriminate between affinities at the high end (e.g., 109M-1-1010M-1) challenges the formulation of a mathematical model able to robustly separate these affinity-dependent responses. Queuing theory enables the analysis of many related processes, such as those resulting from the stochasticity of protein binding and unbinding events. Here we define a network of queues, consisting of BCR early signaling states and transition rates related to the propensity of molecular aggregates to form or disassemble. By considering the family of marginal distributions of BCRs in a given signaling state, we report a significant separation (measured as Jensen-Shannon divergence) that arises from a broad spectrum of antigen affinities.

  14. Network-of-queues approach to B-cell-receptor affinity discrimination.

    PubMed

    Felizzi, Federico; Comoglio, Federico

    2012-06-01

    The immune system is one of the most complex signal processing machineries in biology. The adaptive immune system, consisting of B and T lymphocytes, is activated in response to a large spectrum of pathogen antigens. B cells recognize and bind the antigen through B-cell receptors (BCRs) and this is fundamental for B-cell activation. However, the system response is dependent on BCR-antigen affinity values that span several orders of magnitude. Moreover, the ability of the BCR to discriminate between affinities at the high end (e.g., 10^{9}M^{-1}-10^{10}M^{-1}) challenges the formulation of a mathematical model able to robustly separate these affinity-dependent responses. Queuing theory enables the analysis of many related processes, such as those resulting from the stochasticity of protein binding and unbinding events. Here we define a network of queues, consisting of BCR early signaling states and transition rates related to the propensity of molecular aggregates to form or disassemble. By considering the family of marginal distributions of BCRs in a given signaling state, we report a significant separation (measured as Jensen-Shannon divergence) that arises from a broad spectrum of antigen affinities. PMID:23005146

  15. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  16. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice*

    PubMed Central

    Bartoi, Tudor; Rigbolt, Kristoffer T. G.; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-01-01

    GABAB receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABAB1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABAB receptors. The transgenic mice express GABAB1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABAB1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABAB receptors via the GABAB2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABAB2. The mice equipped with tags on GABAB1 facilitate validation and identification of native binding partners of GABAB receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  17. GABAB receptor constituents revealed by tandem affinity purification from transgenic mice.

    PubMed

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-07-01

    GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  18. Affinity capture of [Arg8]vasopressin-receptor complex using immobilized antisense peptide.

    PubMed Central

    Lu, F X; Aiyar, N; Chaiken, I

    1991-01-01

    Solubilized noncovalent complexes of [Arg8]-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor [pro-AVP/BNPII-(20-1)], a region that includes the AVP sequence at residues 1-9. The AVP-related AS peptide previously was shown to bind selectively to AVP. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarily at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, [3H]AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. When this fraction was eluted through AS pro-AVP/BNPII-(20-1) silica, a complex containing [3H]AVP was bound and separated from the major, unretarded membrane protein fraction as well as from free AVP. Chemical crosslinking of [3H]AVP complex, SDS/PAGE of the products, and analysis of gel slices by scintillation counting led to detection of two major radiolabeled bands of 31 and 38 kDa. Covalent labeling was blocked when unlabeled AVP was added as a competitor before crosslinking. A third radiolabeled protein band of 15 kDa was found when the receptor complex was solubilized from rat liver membranes in the absence of the protease inhibitor phenylmethylsulfonyl fluoride. Covalently crosslinked [3H]AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photo-affinity labeling, we conclude

  19. Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: synthesis, structure-affinity relationships and molecular modeling studies.

    PubMed

    Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Poli, Daniela; Squarcialupi, Lucia; Filacchioni, Guido; Varani, Katia; Vincenzi, Fabrizio; Borea, Pier Andrea; Dal Ben, Diego; Lambertucci, Catia; Cristalli, Gloria

    2013-01-01

    A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (hetero)aryl moiety (compounds 1-8) but also a carboxylate group (9-14), were designed as hA(3) AR antagonists. This study produced some interesting compounds endowed with good hA(3) receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivatives (series B-2) do not bind or bind with very low affinity at the hA(3) AR, the only exception being the 5-N-benzoyl compound 19 that shows a hA(3)K(i) value in the high μ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles 20-24 with modest affinity but high selectivity toward the hA(3) AR subtype. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA(3) receptor. PMID:23171656

  20. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  1. The bovine peripheral-type benzodiazepine receptor: A receptor with low affinity for benzodiazepines

    SciTech Connect

    Parola, A.L.; Laird, H.E. II )

    1991-01-01

    The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit ({sup 3}H)PK 11195 binding was PK 11195 > protoporphyrin IX > benzodiazepines. ({sup 3}H)PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. ({sup 3}H)PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing and denaturing conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.

  2. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    NASA Astrophysics Data System (ADS)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  3. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    PubMed

    Cozzi, Nicholas V; Daley, Paul F

    2016-02-01

    N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated

  4. Binding characteristics of [3H]14-methoxymetopon, a high affinity mu-opioid receptor agonist.

    PubMed

    Spetea, Mariana; Tóth, Fanni; Schütz, Johannes; Otvös, Ferenc; Tóth, Géza; Benyhe, Sandor; Borsodi, Anna; Schmidhammer, Helmut

    2003-07-01

    The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level. PMID:12887410

  5. Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods.

    PubMed

    Roos, Katarina; Hogner, Anders; Ogg, Derek; Packer, Martin J; Hansson, Eva; Granberg, Kenneth L; Evertsson, Emma; Nordqvist, Anneli

    2015-12-01

    In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R(2) = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R(2) = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures. PMID:26572910

  6. High-Affinity Fc Receptor Expression Indicates Relative Immaturity in Human Monocytes.

    PubMed

    Clanchy, Felix I L

    2016-05-01

    Within monocyte heterogeneity, subsets represent discrete, well-characterized phenotypes. Although many studies have highlighted differences between subsets, there is evidence that subpopulations represent contiguous stages in a maturational series. As CD14(hi)CD64(hi) monocytes have higher proliferative potential than CD14(hi)CD64(lo) monocytes, the surface marker profile on 4 subsets defined by CD14 and CD64 was measured. The profiles were compared to that of subsets defined by the high-affinity IgE receptor (FcɛRIα), CD16, and CD14; further differences in size, granularity, and buoyancy were measured in subsets delineated by these markers. There was a positive correlation between proliferative monocyte (PM) prevalence and CD64 expression on the classical monocyte subset, and also between PM prevalence and circulating FcɛRIα(+) monocytes. The expression of CD64, the high-affinity IgG receptor, on canonical human monocyte subsets was determined before and after short-term culture, and in response to interleukin (IL)-6, IL-10, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and interferon-γ; the influence of these cytokines on monocyte subset transition was also measured. The loss of FcɛRIα expression preceded an increase in CD16 expression in whole blood cultures. These data indicate that high-affinity Fc receptors are expressed on less mature monocytes and that FcɛRIα(+) monocytes are developmentally antecedent to the canonical classical and intermediate monocyte subsets. PMID:26714112

  7. Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

    PubMed Central

    Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

    1998-01-01

    The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding

  8. Synthesis, structure-affinity relationships, and radiolabeling of selective high-affinity 5-HT4 receptor ligands as prospective imaging probes for positron emission tomography.

    PubMed

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L; Pike, Victor W

    2010-10-14

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT(4) receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT(4) antagonist (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron emitter, either carbon-11 (t(1/2) = 20.4 min) or fluorine-18 (t(1/2) = 109.7 min), and included (i) replacement of the iodine atom with a small substituent such as nitrile, methyl, or fluoro, (ii) methylation of the 8-amino group, (iii) opening of the dioxan ring, and (iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT(4) receptors with amenability to labeling with a positron emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-(11)C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [(11)C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT(4) receptors. PMID:20812727

  9. Synthesis, Structure-affinity Relationships and Radiolabeling of Selective High-affinity 5-HT4 Receptor Ligands as Prospective Imaging Probes for PET

    PubMed Central

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L.; Pike, Victor W.

    2010-01-01

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT4 receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT4 antagonist, (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron-emitter, either carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.7 min), and included, i) replacement of the iodine atom with a small substituent such as nitrile, methyl or fluoro, ii) methylation of the 8-amino group, iii) opening of the dioxan ring, and iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT4 receptors with amenability to labeling with a positron-emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-11C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [11C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT4 receptors. PMID:20812727

  10. Human CD4+ T-Cells: A Role for Low-Affinity Fc Receptors

    PubMed Central

    Chauhan, Anil K.

    2016-01-01

    Both lymphoid and myeloid cells express Fc receptors (FcRs). Low-affinity FcRs engage circulating immune complexes, which results in the cellular activation and pro-inflammatory cytokine production. FcRs participate in the internalization, transport, and/or recycling of antibodies and antigens. Cytosolic FcRs also route these proteins to proteasomes and antigen-presentation pathways. Non-activated CD4+ T-cells do not express FcRs. Once activated, naive CD4+ T-cells express FcγRIIIa, which, upon IC ligation, provide a costimulatory signal for the differentiation of these cells into effector cell population. FcγRIIIa present on CD4+ T-cell membrane could internalize nucleic acid-containing ICs and elicit a cross-talk with toll-like receptors. FcγRIIIa common γ-chain forms a heterodimer with the ζ-chain of T-cell receptor complex, suggesting a synergistic role for these receptors. This review first summarizes our current understanding of FcRs on CD4+ T-cells. Thereafter, I will attempt to correlate the findings from the recent literature on FcRs and propose a role for these receptors in modulating adaptive immune responses via TLR signaling, nucleic acid sensing, and epigenetic changes in CD4+ T-cells. PMID:27313579

  11. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    SciTech Connect

    Alcaraz, G.; Kinet, J.P.; Liu, T.Y.; Metzger, H.

    1987-05-05

    The ..cap alpha.., ..beta.., ..gamma.. subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the ..cap alpha.. chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for ..cap alpha.. and ..beta... However, these putative homologies were not apparent when tryptic maps of the biosynthetically ((/sup 3/H)leucine) labeled subunits were analyzed.

  12. Lack of dopamine D4 receptor affinity contributes to the procognitive effect of lurasidone.

    PubMed

    Murai, Takeshi; Nakako, Tomokazu; Ikeda, Kazuhito; Ikejiri, Masaru; Ishiyama, Takeo; Taiji, Mutsuo

    2014-03-15

    We previously demonstrated among several antipsychotics exhibiting potent dopamine D2 receptor antagonism that only lurasidone, (1R,2S,3R,4S)-N-[(1R,2R)-2-[4-(1,2-benzisothiazol-3-yl)-1-piperazinylmethyl]-1-cyclohexylmethyl]-2,3-bicyclo[2.2.1] heptanedicarboximide hydrochloride, improved performance in the object retrieval detour (ORD) task by marmosets. The mechanisms by which only lurasidone causes enhancements in cognitive function have not yet been established; however, most antipsychotics, except for lurasidone, have been shown to exhibit potent antagonistic activity against the dopamine D4 receptor. The objectives of this study were to evaluate the role of the dopamine D4 receptor on executive function with the selective agonist, Ro10-5824 and antagonist, L-745,870, and elucidate a possible mechanism for the procognitive effect of lurasidone. The effects of these drugs were evaluated in naïve marmosets using the ORD task. Changes in the success rate during the difficult trial in the task were used to assess the cognitive effect of the drugs. Ro10-5824 (0.3-3 mg/kg) increased the success rate in the difficult trial, potentiated the effect of lurasidone, and reversed the cognitive impairment induced by clozapine. Interestingly, the co-administration of L-745,870 with lurasidone decreased the success rate in the difficult trial, whereas the single administration of L-745,870 had no effect. These results suggest that activation of the dopamine D4 receptor may improve executive function, whereas concomitant blockade of dopamine D4 and D2 receptors may have the opposite effect. In addition to the other unique binding profiles of other monoamine receptors, the lack of affinity for the dopamine D4 receptor by lurasidone could also contribute, at least partly, to its cognitive-enhancing effect. PMID:24304719

  13. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    PubMed

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  14. Synthesis, structure-affinity relationships, and molecular modeling studies of novel pyrazolo[3,4-c]quinoline derivatives as adenosine receptor antagonists.

    PubMed

    Lenzi, Ombretta; Colotta, Vittoria; Catarzi, Daniela; Varano, Flavia; Squarcialupi, Lucia; Filacchioni, Guido; Varani, Katia; Vincenzi, Fabrizio; Borea, Pier Andrea; Dal Ben, Diego; Lambertucci, Catia; Cristalli, Gloria

    2011-06-15

    This paper reports the study of new 2-phenyl- and 2-methylpyrazolo[3,4-c]quinolin-4-ones (series A) and 4-amines (series B), designed as adenosine receptor (AR) antagonists. The synthesized compounds bear at the 6-position various groups, with different lipophilicity and steric hindrance, that were thought to increase human A(1) and A(2A) AR affinities and selectivities, with respect to those of the parent 6-unsubstituted compounds. In series A, this modification was not tolerated since it reduced AR affinity, while in series B it shifted the binding towards the hA(1) subtype. To rationalize the observed structure-affinity relationships, molecular docking studies at A(2A)AR-based homology models of the A(1) and A(3) ARs and at the A(2A)AR crystal structure were carried out. PMID:21616671

  15. The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

    PubMed Central

    Uehara, Hirotsugu; Hocart, Simon J.; González, Nieves; Mantey, Samuel A.; Nakagawa, Tomoo; Katsuno, Tatsuro; Coy, David H.; Jensen, Robert T.

    2012-01-01

    There is increased interest in the Bn-receptor family because they are frequently over/ectopically-expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog,[D-Tyr6,β-Ala11,Phe13,Nle14]Bn(6-14)[Univ.Lig] has the unique property of having high affinity for all three human BNRs(GRPR,NMBR,BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor(CCKAR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCKAR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig(>2-fold) with 2 in EC2[ D97N,G112V], 1 in UTM6[Y284A], 2 in EC4[R287N,H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-pi or H–bonding interactions are also important for determining its high affinity. PMID:22828605

  16. Regulator of insulin receptor affinity in rat skeletal muscle sarcolemmal vesicles

    SciTech Connect

    Whitson, R.H.; Barnard, K.J.; Kaplan, S.A.; Itakura, K.

    1986-05-01

    Wheat germ agglutinin (WGA) affinity purification of detergent solubilized insulin receptors (IR) from rat skeletal muscle sarcolemmal vesicles resulted in an apparent increase in total insulin binding activity of greater than 5-fold, suggesting that an inhibitory component had been removed. This was verified when the flow-through fraction from the WGA column was dialyzed and added back to the partially purified receptor. The addition of a 100-fold dilution of the inhibitor preparation caused a 50% reduction in binding to trace amounts of /sup 125/I-insulin. Scatchard analysis revealed that the effect of the inhibitor was to decrease the affinity of the muscle IR. The inhibitor appeared to be tissue specific, inasmuch as the I/sub 50/'s for WGA-purified IR from rat fat and liver were 10 times the I/sub 50/ for muscle IR. The I/sub 50/ for insulin binding to intact IM-9 cells was 30 times the value for muscle IR. The inhibitor eluted in the void volume of Sephadex G-50 columns. Its activity was not destroyed by heating at 90/sup 0/C for 10 minutes, or by prolonged incubation with trypsin or dithiothreitol. The inhibitor described here may have a role in modulating insulin sensitivity in skeletal muscle.

  17. Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment*

    PubMed Central

    Kofoed, Eric M.; Guerbadot, Martin; Schaufele, Fred

    2010-01-01

    An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor α-isoform (ERα) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ERα with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ERαs at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ERα monomer at higher SRC-RID concentrations (∼10 nm). The methods also showed that only a subpopulation of ERα was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment. PMID:19926790

  18. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  19. Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

    SciTech Connect

    Hass, P.E.; Hotchkiss, A.; Mohler, M.; Aggarwal, B.B.

    1985-10-05

    Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

  20. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

    PubMed

    Kessler, R M; Ansari, M S; de Paulis, T; Schmidt, D E; Clanton, J A; Smith, H E; Manning, R G; Gillespie, D; Ebert, M H

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8. PMID:1831229

  1. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides

    SciTech Connect

    Kessler, R.M.; Ansari, M.S.; de Paulis, T.; Schmidt, D.E.; Clanton, J.A.; Smith, H.E.; Manning, R.G.; Gillespie, D.; Ebert, M.H. )

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. Iodopride (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with (125I)iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, iclopride (KD 0.23 nM) and itopride (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, epidepride (KD 0.057 nM) and ioxipride (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

  2. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  3. Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

    PubMed

    Di Tomasso, Geneviève; Salvail-Lacoste, Alix; Bouvette, Jonathan; Omichinski, James G; Legault, Pascale

    2014-01-01

    Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest. PMID:25432744

  4. Towards a high-affinity allosteric enhancer at muscarinic M1 receptors.

    PubMed

    Lazareno, Sebastian; Popham, Angela; Birdsall, Nigel J M

    2002-01-01

    Loss of forebrain acetylcholine (ACh) is an early neurochemical lesion in Alzheimer's Disease (AD), and muscarinic receptors for ACh are involved in memory and cognition, so a muscarinic agonist could provide 'replacement therapy' in this disease. Muscarinic receptors, which couple to G-proteins, occur throughout the CNS, and in the periphery they mediate the responses of the parasympathetic nervous system, so selectivity is crucial. The five subtypes of muscarinic receptor, M1-M5, have a distinct regional distribution, with M2 and M3 mediating most of the peripheral effects, M2 predominating in hindbrain areas, and M1 predominating in the cortex and hippocampus--the brain regions most associated with memory and cognition, which has lead to a search for a truly M1-selective muscarinic agonist. That search has so far been unsuccessful, but acetylcholinesterase inhibitors such as donepezil (Aricept), which potentiate cholinergic neurotransmission, have a therapeutic role in the management of AD; so the M1 receptor remains a therapeutic target. Our approach is to develop allosteric enhancers--compounds which bind to the receptor at an 'allosteric' site which is distinct from the 'primary' site to which the endogenous ligand binds, and which enhance the affinity (or efficacy) of the endogenous ligand. We have developed radioligand binding assays and analyses for the detection and quantitatitation of allosteric interactions of a test agent with labelled and unlabelled 'primary' ligands, and we report here some results of the initial phase of a chemical synthesis project to develop potent and selective allosteric enhancers at muscarinic M1 receptors. PMID:12212769

  5. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    SciTech Connect

    Gil, D.W.; Wolfe, B.B.

    1986-05-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands (/sup 3/H)quinuclidinyl benzilate or (/sup 3/H)PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of (/sup 3/H)quinuclidinyl benzilate in a biphasic manner.

  6. Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

    SciTech Connect

    Husman, B.; Haldosen, L.A.; Andersson, G.; Gustafsson, J.A.

    1988-03-15

    Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H/sub 2/O and D/sub 2/O, and affinity cross-linking using /sup 125/I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of /sup 125/I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble /sup 125/I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the /sup 125/I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity.

  7. Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists

    PubMed Central

    Li, An-Hu; Moro, Stefano; Melman, Neli; Ji, Xiao-duo; Jacobson, Kenneth A.

    2012-01-01

    The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure–activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore

  8. Identification and function of adenosine A3 receptor in afferent arterioles.

    PubMed

    Lu, Yan; Zhang, Rui; Ge, Ying; Carlstrom, Mattias; Wang, Shaohui; Fu, Yiling; Cheng, Liang; Wei, Jin; Roman, Richard J; Wang, Lei; Gao, Xichun; Liu, Ruisheng

    2015-05-01

    Adenosine plays an important role in regulation of renal microcirculation. All receptors of adenosine, A1, A2A, A2B, and A3, have been found in the kidney. However, little is known about the location and function of the A3 receptor in the kidney. The present study determined the expression and role of A3 receptors in mediating the afferent arteriole (Af-Art) response and studied the interaction of A3 receptors with angiotensin II (ANG II), A1 and A2 receptors on the Af-Art. We found that the A3 receptor expressed in microdissected isolated Af-Art and the mRNA levels of A3 receptor were 59% of A1. In the isolated microperfused Af-Art, A3 receptor agonist IB-MECA did not have a constrictive effect. Activation of A3 receptor dilated the preconstricted Af-Art by norepinephrine and blunted the vasoconstrictive effect of both adenosine A1 receptor activation and ANG II on the Af-Art, respectively. Selective A2 receptor antagonist (both A2A and A2B) had no effect on A3 receptor agonist-induced vasodilation, indicating that the dilatory effect of A3 receptor activation is not mediated by activation of A2 receptor. We conclude that the A3 receptor is expressed in the Af-Art, and activation of the A3 receptor dilates the Af-Art. PMID:25608966

  9. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1989-12-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.

  10. Regulation of low affinity neurotrophin receptor (p75NTR) by early growth response (Egr) transcriptional regulators

    PubMed Central

    Gao, Xiaoguang; Daugherty, Rebecca L.; Tourtellotte, Warren G.

    2007-01-01

    The low affinity neurotrophin receptor p75NTR is a multifunctional receptor with important roles in neurotrophin signaling, axon outgrowth, and oligodendroglia and neuron survival. It is transcriptionally regulated with spatial and temporal precision during nervous system development, injury and regeneration. Very little is known about how p75NTR expression is dynamically regulated but it is likely to influence how p75NTR signals in particular cellular contexts. Here, we identify the early growth response (Egr) transcriptional regulators, Egr1 and Egr3, as direct modulators of p75NTR gene expression. Egr1 and Egr3 bind and transactivate the p75NTR promoter in vitro and in vivo, using distinct response elements on the p75NTR promoter. Consistent with these results, p75NTR expression is greatly diminished in muscle spindle stretch receptors and in peripheral nerve Schwann cells in Egr gene deficient mice. Taken together, the results elucidate a novel mechanism whereby Egr proteins can directly modulate p75NTR expression and signaling in vivo. PMID:17916431

  11. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    PubMed

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. PMID:27100851

  12. The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes.

    PubMed

    Roffel, A F; in't Hout, W G; de Zeeuw, R A; Zaagsma, J

    1987-05-01

    We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepine exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 microM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors. PMID:3614390

  13. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  14. Structure-affinity relationships of halogenated predicentrine and glaucine derivatives at D1 and D2 dopaminergic receptors: halogenation and D1 receptor selectivity.

    PubMed

    Asencio, Marcelo; Hurtado-Guzmán, Claudio; López, John J; Cassels, Bruce K; Protais, Philippe; Chagraoui, Abdeslam

    2005-06-01

    Halogenation of the aporphine alkaloid boldine at the 3-position leads to increased affinity for rat brain D(1)-like dopaminergic receptors with some selectivity over D(2)-like receptors. A series of 3-halogenated and 3,8-dihalogenated (halogen=Cl, Br or I) derivatives of predicentrine (9-O-methylboldine) and glaucine (2,9-di-O-methylboldine) were prepared and assayed for binding at D(1) and D(2) sites. Halogenation of predicentrine led to strong increases in affinity for D(1)-like receptors, while the affinities for D(2)-like receptors were either practically unchanged or reduced three- to fourfold. Halogenated glaucine derivatives did not show any clear trend towards enhanced selectivity, and the affinities were poor and similar to or worse than the values previously recorded for glaucine itself. Together with earlier work on boldine derivatives, these results suggest that the 2-hydroxy group on the aporphine skeleton may determine a binding mode favoring D(1)-like over D(2)-like receptors, with enhanced affinity when the C-3 position is halogenated. PMID:15862999

  15. Role of the High Affinity Immunoglobulin E Receptor in Bacterial Translocation and Intestinal Inflammation

    PubMed Central

    Dombrowicz, David; Nutten, Sophie; Desreumaux, Pierre; Neut, Christel; Torpier, Gérard; Peeters, Marc; Colombel, Jean-Frédéric; Capron, Monique

    2001-01-01

    A role for immunoglobulin E and its high affinity receptor (FcεRI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized FcεRI (hFcεRI), with a cell distribution similar to that in humans, to FcεRI-deficient animals. In hFcεRI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFcεRI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas FcεRI-deficient animals were protected from colitis, demonstrating that FcεRI can affect the onset of intestinal inflammation. PMID:11136818

  16. Glycan-based high-affinity ligands for toxins and pathogen receptors.

    PubMed

    Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S

    2010-03-01

    Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan-based high-affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the "glycocode" and how these glycans participate in normal and aberrant cellular communication. PMID:20135686

  17. Can thermodynamic measurements of receptor binding yield information on drug affinity and efficacy?

    PubMed

    Borea, P A; Dalpiaz, A; Varani, K; Gilli, P; Gilli, G

    2000-12-01

    The present commentary surveys the methods for obtaining the thermodynamic parameters of the drug-receptor binding equilibrium, DeltaG degrees, DeltaH degrees, DeltaS degrees, and DeltaC degrees (p) (standard free energy, enthalpy, entropy, and heat capacity, respectively). Moreover, it reviews the available thermodynamic data for the binding of agonists and antagonists to several G-protein coupled receptors (GPCRs) and ligand-gated ion channel receptors (LGICRs). In particular, thermodynamic data for five GPCRs (beta-adrenergic, adenosine A(1), adenosine A(2A), dopamine D(2), and 5-HT(1A)) and four LGICRs (glycine, GABA(A), 5-HT(3), and nicotinic) have been collected and analyzed. Among these receptor systems, seven (three GPCRs and all LGICRs) show "thermodynamic agonist-antagonist discrimination": when the agonist binding to a given receptor is entropy-driven, the binding of its antagonist is enthalpy-driven, or vice versa. A scatter plot of all entropy versus enthalpy values of the database gives a regression line with the equation TDeltaS degrees (kJ mol(-1); T = 298.15 K) = 40.3 (+/- 0.7) + 1.00 (+/-0.01) DeltaH degrees (kJ mol(-1)); N = 184; r = 0.981; P < 0.0001 - which is of the form DeltaH degrees = beta. DeltaS degrees, revealing the presence of the "enthalpy-entropy compensation" phenomenon. This means that any decrease of binding enthalpy is compensated for by a parallel decrease of binding entropy, and vice versa, in such a manner that affinity constant values (K(A)) of drug-receptor equilibrium (DeltaG degrees = -RT ln K(A) = DeltaH degrees - TDeltaS degrees ) cannot be greater than 10(11) M(-1). According to the most recent hypotheses concerning drug-receptor interaction mechanisms, these thermodynamic phenomena appear to be a consequence of the rearrangement of solvent molecules that occurs during the binding. PMID:11077036

  18. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  19. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  20. A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor

    PubMed Central

    Jun, Lucy S.; Showalter, Aaron D.; Ali, Nosher; Dai, Feihan; Ma, Wenzhen; Coskun, Tamer; Ficorilli, James V.; Wheeler, Michael B.; Michael, M. Dodson; Sloop, Kyle W.

    2014-01-01

    Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r−/− animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner

  1. Novel potent and highly selective human A(3) adenosine receptor antagonists belonging to the 4-amido-2-arylpyrazolo[3,4-c]quinoline series: molecular docking analysis and pharmacological studies.

    PubMed

    Colotta, Vittoria; Capelli, Francesca; Lenzi, Ombretta; Catarzi, Daniela; Varano, Flavia; Poli, Daniela; Vincenzi, Fabrizio; Varani, Katia; Borea, Pier Andrea; Dal Ben, Diego; Volpini, Rosaria; Cristalli, Gloria; Filacchioni, Guido

    2009-01-01

    The study of novel 2-arylpyrazolo[3,4-c]quinolin-4-(hetero)arylamides, designed as human (h) A(3) adenosine receptor antagonists, is reported. The new derivatives are endowed with nanomolar hA(3) receptor affinity and high selectivity versus hA(1), hA(2A) and hA(2B) receptors. Among the (hetero)aroyl residues introduced on the 4-amino group, the 2-furyl and 4-pyridyl rings turned out to be the most beneficial for hA(3) affinity (K(i)=3.4 and 5.0nM, respectively). An intensive molecular docking study to a rhodopsin-based homology model of the hA(3) receptor was carried out to obtain a 'structure-based pharmacophore model' that proved to be helpful for the interpretation of the observed affinities of the new hA(3) pyrazoloquinoline antagonists. PMID:18996701

  2. Synthesis and opioid receptor affinity of a series of 2, 4-diaryl-substituted 3,7-diazabicylononanones.

    PubMed

    Siener, T; Cambareri, A; Kuhl, U; Englberger, W; Haurand, M; Kögel, B; Holzgrabe, U

    2000-10-01

    3,7-Diazabicyclo[3.3.1]nonan-9-ones having aryl rings in positions 2 and 4 with systematically varied substituents were synthesized using a double Mannich procedure. Radioligand binding assays were performed to measure the affinity of the compounds to the mu-, delta-, and kappa-opioid receptors. The affinity of all 2, 4-diphenyl-substituted 3,7-diazabicyclo[3.3.1]nonan-9-ones to the mu- and delta-receptors was found to be low. In contrast, with exception of the nitro- and cyanophenyl-substituted compounds, most of the diazabicycles showed considerable affinity for the kappa-receptor. In particular, the m-fluoro-, p-methoxy-, and m-hydroxy-substituted compounds have an affinity in the submicromolar range. Due to solubility problems in aqueous media, salts of HZ2 were synthesized. The methiodide shows high kappa-affinity and may, thus, be a promising candidate for development of a peripheral kappa-agonist, e.g. for use in the case of rheumatoid arthritis. PMID:11020289

  3. Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

    PubMed Central

    Piper, J W; Swerlick, R A; Zhu, C

    1998-01-01

    Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5). PMID:9449350

  4. Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

    PubMed

    Bozon, V; Pajot-Augy, E; Vignon, X; Salesse, R

    1998-08-25

    In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars. PMID:9806348

  5. New ursane triterpenoids from Ficus pandurata and their binding affinity for human cannabinoid and opioid receptors.

    PubMed

    Khedr, Amgad I M; Ibrahim, Sabrin R M; Mohamed, Gamal A; Ahmed, Hany E A; Ahmad, Amany S; Ramadan, Mahmoud A; El-Baky, Atef E Abd; Yamada, Koji; Ross, Samir A

    2016-07-01

    Phytochemical investigation of Ficus pandurata Hance (Moraceae) fruits has led to the isolation of two new triterpenoids, ficupanduratin A [1β-hydroxy-3β-acetoxy-11α-methoxy-urs-12-ene] (11) and ficupanduratin B [21α-hydroxy-3β-acetoxy-11α-methoxy-urs-12-ene] (17), along with 20 known compounds: α-amyrin acetate (1), α-amyrin (2), 3β-acetoxy-20-taraxasten-22-one (3), 3β-acetoxy-11α-methoxy-olean-12-ene (4), 3β-acetoxy-11α-methoxy-12-ursene (5), 11-oxo-α-amyrin acetate (6), 11-oxo-β-amyrin acetate (7), palmitic acid (8), stigmast-4,22-diene-3,6-dione (9), stigmast-4-ene-3,6-dione (10), stigmasterol (12), β-sitosterol (13), stigmast-22-ene-3,6-dione (14), stigmastane-3,6-dione (15), 3β,21β-dihydroxy-11α-methoxy-olean-12-ene (16), 3β-hydroxy-11α-methoxyurs-12-ene (18), 6-hydroxystigmast-4,22-diene-3-one (19), 6-hydroxystigmast-4-ene-3-one (20), 11α,21α-dihydroxy-3β-acetoxy-urs-12-ene (21), and β-sitosterol-3-O-β-D-glucopyranoside (22). Compound 21 is reported for the first time from a natural source. The structures of the 20 compounds were elucidated on the basis of IR, 1D ((1)H and (13)C), 2D ((1)H-(1)H COSY, HSQC, HMBC and NOESY) NMR and MS spectroscopic data, in addition to comparison with literature data. The isolated compounds were evaluated for their anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities. In addition, their radioligand displacement affinity on opioid and cannabinoid receptors was assessed. Compounds 4, 11, and 15 exhibited good affinity towards the CB2 receptor, with displacement values of 69.7, 62.5 and 86.5 %, respectively. Furthermore, the binding mode of the active compounds in the active site of the CB2 cannabinoid receptors was investigated through molecular modelling. PMID:27350550

  6. Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

    PubMed Central

    Pierce, E J; Davison, M D; Parton, R G; Habig, W H; Critchley, D R

    1986-01-01

    Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. Images Fig. 5. PMID:3539106

  7. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  8. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    SciTech Connect

    Sherman, S.J.; Catterall, W.A.

    1982-11-01

    Specific binding of /sup 3/H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors.

  9. Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

    PubMed

    Aoshima, H; Inoue, Y; Hori, K

    1992-10-01

    Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1337082

  10. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    PubMed

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases. PMID:26297614

  11. Gestational treatment with cocaine and fluoxetine alters oxytocin receptor number and binding affinity in lactating rat dams.

    PubMed

    Johns, Josephine M; Lubin, Deborah A; Walker, Cheryl H; Joyner, Paul; Middleton, Christopher; Hofler, Vivian; McMurray, Matthew

    2004-01-01

    Cocaine administered chronically throughout gestation has been correlated with deficits in maternal behavior, increased maternal aggressive behavior and decreased oxytocin levels in rats. In addition to its effects on oxytocin levels, cocaine is a potent serotonergic, dopaminergic and noradrenergic reuptake inhibitor. Alterations in the dopaminergic and serotonergic systems have been suggested as possibly having a role in cocaine-induced maternal aggression. This study was in part, an attempt to understand some of the mechanisms by which cocaine increases postpartum aggression, particularly as they relate to changes in the oxytocin system. Oxytocin receptor number and binding affinity in the medial preoptic area of the hypothalamus, ventral tegmental area, hippocampus and amygdala were determined for lactating rat dams on postpartum day 6 (PPD 6) that were gestationally treated with cocaine, fluoxetine, saline or an amfonelic acid/fluoxetine drug combination. Cocaine and fluoxetine treatment both resulted in a significant up-regulation of oxytocin receptor number and lower receptor affinity in the amygdala of lactating rat dams compared to saline controls and the amfonelic acid/fluoxetine combination treatment group. Cocaine treatment also resulted in a significant down-regulation of oxytocin receptors in the medial preoptic area and both cocaine and fluoxetine treated dams had the highest affinity for oxytocin receptors in this brain region. Results of the present study support previous data indicating that alterations in oxytocinergic and perhaps serotonergic system dynamics in the amygdala may play a role in cocaine-induced postpartum aggression. PMID:15380831

  12. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  13. Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

    PubMed

    Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

    2014-01-01

    Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] PMID:25311884

  14. Cardiac and gastric effects of histamine H2 receptor antagonists: no evidence for a correlation between lipophilicity and receptor affinity.

    PubMed Central

    Coruzzi, G.; Adami, M.; Pozzoli, C.; Giorgi, F.; Bertaccini, G.

    1996-01-01

    1. A series of histamine H2 receptor antagonists with different lipophilicity were tested in cardiac and gastric assays in order to reveal possible differences in receptor affinity. Lipophilicity of the compounds was expressed as CLOG P (theoretically-determined logarithm of octanol:water partition coefficient) and log k' (logarithm of capacity factor, experimentally-determined by reverse-phase high performance liquid chromatography). 2. Aminopotentidine (APT) and iodoaminopotentidine (I-APT), which are both lipophilic compounds, behaved as insurmountable antagonists of histamine responses in rat isolated gastric fundus (pKB = 6.20 +/- 0.16 and 6.89 +/- 0.19, respectively) and guinea-pig isolated papillary muscle (pKB = 6.34 +/- 0.37 and 6.81 +/- 0.26, respectively). They were approximately as effective as ranitidine (RAN) in reducing histamine-induced acid secretion in the anaesthetized rat, ID50 values being 0.018 +/- 0.02, 0.020 +/- 0.03 and 0.036 +/- 0.01 mumol kg-1 i.v. for APT, I-APT and RAN, respectively. Both APT and I-APT had a significantly longer duration of action than RAN. 3. The hydrophilic compound, SK&F 92857, was inactive up to 10 microM in modifying histamine-induced acid secretion in the isolated rat stomach. In the papillary muscle, low concentrations (0.1-1 microM) of this compound produced a competitive antagonism of the histamine responses (pA2 value = 7.38 +/- 0.11), while a higher concentration (10 microM) significantly reduced the maximal response to histamine. 4. RAN competitively antagonized histamine effects with a comparable affinity in cardiac and gastric preparations (pA2 values were 6.42 +/- 0.09 and 6.78 +/- 0.38 in heart and stomach, respectively). 5. Results obtained in this study clearly showed that the discrepancies between gastric and cardiac effects observed for some H2 antagonists are not explained solely by differences in lipophilicity of compounds. Moreover, the significant correlation found between CLOG P and log k

  15. Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

    SciTech Connect

    Zhang, Q.Y.; Menon, K.M.J. )

    1989-11-01

    Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for {sup 125}I-labeled human choriogonadotropin, with an equilibrium dissociation constant (K{sub d}) of 2.5 {times} 10{sup {minus}9} M, which is comparable to the K{sub d} values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the M{sub r} 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified M{sub r} 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the M{sub r} 135,000 species was almost completely converted to a M{sub r} 92,000 band, but the M{sub r} 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked M{sup r} 92,000 subunit, which comprises the hormone-binding domain.

  16. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  17. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    PubMed

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-02-01

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands. PMID:24476714

  18. Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta.

    PubMed

    Nagai, Taku; Furusho, Yuko; Li, Hua; Hasui, Kazuhisa; Matsukita, Sumika; Sueyoshi, Kazunobu; Yanagi, Masakazu; Hatae, Masaki; Takao, Sonshin; Matsuyama, Takami

    2015-06-01

    Folate receptors α (FRα) and β (FRβ) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRα is rare in normal cells and elevated in cancer cells. Thus, FRα-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRβ and that these cells can enhance tumor growth. Although FRα and FRβ share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRα-expressing cancer cells and FRβ-expressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRα or anti-FRβ MAbs. In this study, we developed a MAb that recognizes both FRα and FRβ (anti-FRαβ). The anti-FRαβ specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRαβ bound to soluble forms of the FRα and FRβ proteins with high affinity (KD=6.26×10(-9) M and 4.33×10(-9) M, respectively). In vitro functional analysis of the anti-FRαβ showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRα-expressing and FRβ-expressing cell lines. The anti-FRαβ MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression. PMID:26090596

  19. Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors.

    PubMed

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2008-02-12

    Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Galpha-subunits (Galpha16 or Galphai2) with the kappa-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S3107.33, F3147.37, and I3167.39 to Y3207.43) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S3117.34 and N3267.49 were found to be sensitive in Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR. PMID:18205395

  20. Synthesis and characterization of a cellular membrane affinity chromatography column containing histamine 1 and P2Y1 receptors: A multiple G-protein coupled receptor column

    PubMed Central

    Moaddel, Ruin; Musyimi, Harrison K.; Sanghvi, Mitesh; Bashore, Charlene; Frazier, Chester R.; Khadeer, Mohammad; Bhatia, Prateek; Wainer, Irving W.

    2015-01-01

    A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding. PMID:19608372

  1. Direct measurement of T cell receptor affinity and sequence from naïve antiviral T cells.

    PubMed

    Zhang, Shu-Qi; Parker, Patricia; Ma, Ke-Yue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad M; Wendel, Ben S; Meriwether, Amanda I; Salazar, Mary Alice; Jiang, Ning

    2016-06-01

    T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCRs). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. We introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8(+) T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen-inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high-affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high-affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

  2. Improved binding affinity and interesting selectivities of aminopyrimidine-bearing carbohydrate receptors in comparison with their aminopyridine analogues.

    PubMed

    Lippe, Jan; Seichter, Wilhelm; Mazik, Monika

    2015-12-28

    Due to the problems with the exact prediction of the binding properties of an artificial carbohydrate receptor, the identification of characteristic structural features, having the ability to influence the binding properties in a predictable way, is of high importance. The purpose of our investigation was to examine whether the previously observed higher affinity of 2-aminopyrimidine-bearing carbohydrate receptors in comparison with aminopyridine substituted analogues represents a general tendency of aminopyrimidine-bearing compounds. Systematic binding studies on new compounds consisting of 2-aminopyrimidine groups confirmed such a tendency and allowed the identification of interesting structure-activity relationships. Receptors having different symmetries showed systematic preferences for specific glycosides, which are remarkable for such simple receptor systems. Particularly suitable receptor architectures for the recognition of selected glycosides were identified and represent a valuable base for further developments in this field. PMID:26467387

  3. Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists.

    PubMed

    Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene; O'Connor, Robert; Chen, Zhoumou; Gizewski, Elizabeth; Crane, Steven; Gao, Zhan-Guo; Auchampach, John A; Salvemini, Daniela; Jacobson, Kenneth A

    2016-04-14

    Purine (N)-methanocarba-5'-N-alkyluronamidoriboside A3 adenosine receptor (A3AR) agonists lacking an exocyclic amine resulted from an unexpected reaction during a Sonogashira coupling and subsequent aminolysis. Because the initial C6-Me and C6-styryl derivatives had unexpectedly high A3AR affinity, other rigid nucleoside analogues lacking an exocyclic amine were prepared. Of these, the C6-Me-(2-phenylethynyl) and C2-(5-chlorothienylethynyl) analogues were particularly potent, with human A3AR Ki values of 6 and 42 nM, respectively. Additionally, the C2-(5-chlorothienyl)-6-H analogue was potent and selective at A3AR (MRS7220, Ki 60 nM) and also completely reversed mouse sciatic nerve mechanoallodynia (in vivo, 3 μmol/kg, po). The lack of a C6 H-bond donor while maintaining A3AR affinity and efficacy could be rationalized by homology modeling and docking of these hypermodified nucleosides. The modeling suggests that a suitable combination of stabilizing features can partially compensate for the lack of an exocyclic amine, an otherwise important contributor to recognition in the A3AR binding site. PMID:26890707

  4. G Protein-Coupled Receptors Directly Bind Filamin A with High Affinity and Promote Filamin Phosphorylation

    PubMed Central

    2015-01-01

    Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure–function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR–cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm. PMID:26460884

  5. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  6. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  7. Comparison of three high affinity SPECT radiotracers for the dopamine D2 receptor.

    PubMed

    al-Tikriti, M S; Baldwin, R M; Zea-Ponce, Y; Sybirska, E; Zoghbi, S S; Laruelle, M; Malison, R T; Kung, H F; Kessler, R M; Charney, D S

    1994-02-01

    The regional brain distribution and pharmacological specificity of three high affinity tracers for the dopamine (DA) D2 receptor: [123I]IBF, [123I]epidepride, and [123I]2'-ISP were assessed by SPECT imaging of non-human primates. The ratios of striatal-to-occipital activities at the time of peak striatal uptake were 2.2, 6.3 and 1.7, respectively. From the peak striatal activities, washout rates were 33, 4 and 16%/h for [123I]IBF, [123I]epidepride and [123I]2'-ISP, respectively. The reversibility of the striatal uptake of all three agents was demonstrated by the rapid displacement induced by the dopamine D2 selective antipsychotic agent raclopride, which increased washout rates to 96, 58 and 43%/h. The administration of d-amphetamine, which induces release of dopamine, had no noticeable effect on [123I]epidepride but increased the washout rate of [123I]IBF. These results suggest that, among these three agents, [123I]epidepride is the superior tracer for in vivo displacement studies because of its slow washout and high target-to-background ratios. However, for tracer kinetic modeling, [123I]IBF may be the superior agent because of its early time of peak uptake and its higher target-to-background ratios than [123I]2'-ISP. PMID:9234281

  8. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Rozengurt, E.

    1985-11-01

    Gastrin-releasing peptide (GRP) labeled with /sup 125/I at tyrosine-15 (/sup 125/I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. /sup 125/I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells.

  9. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  10. Roles of the NMDA Receptor and EAAC1 Transporter in the Modulation of Extracellular Glutamate by Low and High Affinity AMPA Receptors in the Cerebellum in Vivo: Differential Alteration in Chronic Hyperammonemia.

    PubMed

    Cabrera-Pastor, Andrea; Taoro, Lucas; Llansola, Marta; Felipo, Vicente

    2015-12-16

    The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors. PMID:26428532

  11. Structure-activity relationships for hallucinogenic N,N-dialkyltryptamines: photoelectron spectra and serotonin receptor affinities of methylthio and methylenedioxy derivatives

    SciTech Connect

    Kline, T.B.; Benington, F.; Morin, R.D.; Beaton, J.M.; Glennon, R.A.; Domelsmith, L.N.; Houk, K.N.; Rozeboom, M.D.

    1982-11-01

    Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity, which may have implications concerning the mechanism of receptor binding.

  12. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    PubMed Central

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCOSTA, Corrie J. B.; Chen, Lin

    2014-01-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding. PMID:23802200

  13. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule.

    PubMed

    Friedman, Mikaela; Orlova, Anna; Johansson, Eva; Eriksson, Tove L J; Höidén-Guthenberg, Ingmarie; Tolmachev, Vladimir; Nilsson, Fredrik Y; Ståhl, Stefan

    2008-03-01

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection. PMID:18207161

  14. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    PubMed

    Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

    2014-11-01

    The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα. PMID:25061691

  15. Effect of prenatal and neonatal exposure to lead on the affinity and number of estradiol receptors in the uterus

    SciTech Connect

    Wiebe, J.P.; Barr, K.J.

    1988-01-01

    Female Sprague-Dawley rats were treated with lead chloride (20 ppm or 200 ppm Pb) or sodium chloride (controls) in their drinking water. Three treatment regimens were employed: (I) rats were treated prior to mating and uteri were removed from 21-d-old offspring, (II) treatments were begun when females were in d 7 of pregnancy and continued on the dams until the pups were 21 d old, and half of these offspring were continued on the Pb treatments and half on saline, with uteri removed during diestrus when female offspring were approximately 150 d old; (III) female rats were treated from d 21 to d 35 and then uteri were removed. Estradiol-receptor binding and affinity were determined on the uterine tissues. Treatment with lead prior to mating (group I) resulted in a significant increase in estradiol-receptor affinity (K/sub a/) in 21-d-old offspring without a change in estradiol receptor number (N). Treatment from d 7 of pregnancy until weaning of the pups resulted in approximately 35% decrease in estradiol receptors per milligram uterine protein when these offspring reached 150 d of age (group II). Similarly, treatment with Pb from d 21 until d 35 or until d 150 resulted in a significant decrease in uterine estradiol receptor number at 35 and 150 d, respectively, while the K/sub a/ was significantly increased by the exposure to Pb. The results demonstrate that prenatal and/or postnatal exposure to PB alters the number and affinity of estradiol receptors in the prepubertal and adult rat uterus.

  16. Current concepts. I. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

    SciTech Connect

    Moody, T.W.; Carney, D.N.; Cuttitta, F.; Quattrocchi, K.; Minna, J.D.

    1985-07-15

    The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (/sup 125/I-Tyr/sup 4/)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated, using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (/sup 125/I-Tyr/sup 4/)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC. 31 references, 6 figures, 2 tables.

  17. Synthesis and Purine Receptor Affinity of 6-Oxopurine Nucleosides and Nucleotides Containing (N)-Methanocarba-pseudoribose Rings

    PubMed Central

    Ravi, Gnana; Lee, Kyeong; Ji, Xiao-duo; Kim, Hak Sung; Soltysiak, Kelly A.; Marquez, Victor E.; Jacobson, Kenneth A.

    2016-01-01

    6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5′-position with an N-methyluronamide or with phosphate groups. PMID:11527718

  18. Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

    PubMed Central

    Ho, Ming-Fen; Low, Leanne M.; Rose’Meyer, Roselyn B.

    2016-01-01

    Background Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A3 receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A3 receptor agonists in coronary blood vessels using the isolated perfused heart preparation. Methods mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A3 receptor mediated coronary vasodilation in the rat heart. Results Adenosine A3 receptor agonists induced coronary vasodilation. The expression of adenosine A3 receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A3 receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A3 receptor agonists. Conclusions This study demonstrated alterations in the expression of adenosine A3 receptors occurred in a tissue specific mode, and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A3 receptor in hypertensive hearts suggest that adenosine A3 receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A3 receptor agonists. PMID:26907173

  19. Reduced secondary cytokine induction by BAY 50-4798, a high-affinity receptor-specific interleukin-2 analog.

    PubMed

    Steppan, Sonja; Eckart, Michael R; Bajsarowicz, Krystyna; Sternberg, Lawrence R; Greve, Jeffrey M; Cassell, Delanie J

    2006-03-01

    Recombinant interleukin-2 (IL-2) (aldesleukin, Proleukin, Chiron, Emeryville, CA) is approved for treatment of cancer patients and under investigation in HIV-infected individuals. However, treatment with aldesleukin is associated with toxicity, which may be due to its elicitation of inflammatory mediators from cells that express the intermediate-affinity IL-2 receptor. BAY 50-4798, a novel IL-2 analog, is a selective agonist for the high-affinity receptor. It induces the proliferation of activated T cells with a potency similar to that of aldesleukin but has reduced activity on cells expressing the intermediate-affinity receptor. In the current study, we compared cytokine responses elicited in peripheral blood mononuclear cell (PBMC) cultures stimulated with BAY 50-4798 or aldesleukin. BAY 50-4798 induced approximately 5-fold lower mean levels of endogenous IL-2 than aldesleukin, and at least 50% lower levels of proinflammatory cytokines, such as tumor necrosis fctor-alpha (TNF-alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma). Furthermore, statistically significant reductions in the levels of IL-5, IL-8, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed in response to BAY 50-4798. These findings increase our understanding of the biologic action of BAY 50-4798 and suggest a mechanism by which it may exhibit better safety than aldesleukin in humans. PMID:16542139

  20. Affinity of Aporphines for the Human 5-HT2A Receptor: Insights from Homology Modeling and Molecular Docking Studies

    PubMed Central

    Pecic, Stevan; Makkar, Pooja; Chaudhary, Sandeep; Reddy, Boojala V.; Navarro, Hernan A.; Harding, Wayne W.

    2010-01-01

    Analogs of nantenine were docked into a modeled structure of the human 5-HT2A receptor using ICM Pro, GLIDE and GOLD docking methods. The resultant docking scores were used to correlate with observed in vitro apparent affinity (Ke) data. The GOLD docking algorithm when used with a homology model of 5-HT2A, based on a bovine rhodopsin template and built by the program MODELLER, gives results which are most in agreement with the in vitro results. Further analysis of the docking poses among members of a C1 alkyl series of nantenine analogs, indicate that they bind to the receptor in a similar orientation, but differently than nantenine. Besides an important interaction between the protonated nitrogen of the C1 alkyl analogs and residue Asp155, we identified Ser242, Phe234 and Gly238 as key residues responsible for the affinity of these compounds for the 5-HT2A receptor. Specifically, the ability of some of these analogs to establish a H-bond with Ser242 and hydrophobic interactions with Phe234 and Gly238 appears to explain their enhanced affinity as compared to nantenine. PMID:20621490

  1. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    SciTech Connect

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  2. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    PubMed

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  3. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors

    PubMed Central

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  4. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

    PubMed

    Kharrat, Riadh; Servent, Denis; Girard, Emmanuelle; Ouanounou, Gilles; Amar, Muriel; Marrouchi, Riadh; Benoit, Evelyne; Molgó, Jordi

    2008-11-01

    Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples. PMID:18990115

  5. Identification of A3 adenosine receptor agonists as novel non-narcotic analgesics.

    PubMed

    Janes, K; Symons-Liguori, A M; Jacobson, K A; Salvemini, D

    2016-04-01

    Chronic pain negatively impacts the quality of life in a variety of patient populations. The current therapeutic repertoire is inadequate in managing patient pain and warrants the development of new therapeutics. Adenosine and its four cognate receptors (A1 , A2A , A2B and A3 ) have important roles in physiological and pathophysiological states, including chronic pain. Preclinical and clinical studies have revealed that while adenosine and agonists of the A1 and A2A receptors have antinociceptive properties, their therapeutic utility is limited by adverse cardiovascular side effects. In contrast, our understanding of the A3 receptor is only in its infancy, but exciting preclinical observations of A3 receptor antinociception, which have been bolstered by clinical trials of A3 receptor agonists in other disease states, suggest pain relief without cardiovascular side effects and with sufficient tolerability. Our goal herein is to briefly discuss adenosine and its receptors in the context of pathological pain and to consider the current data regarding A3 receptor-mediated antinociception. We will highlight recent findings regarding the impact of the A3 receptor on pain pathways and examine the current state of selective A3 receptor agonists used for these studies. The adenosine-to-A3 receptor pathway represents an important endogenous system that can be targeted to provide safe, effective pain relief from chronic pain. PMID:26804983

  6. Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    PubMed Central

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Thomasson, Kathryn A; Porter, James E

    2006-01-01

    Background Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. Results Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant

  7. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    SciTech Connect

    Todd, S.L.; Balster, R.L.; Martin, B.R. )

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  8. Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy µ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities

    PubMed Central

    Harland, Aubrie A.; Yeomans, Larisa; Griggs, Nicholas W.; Anand, Jessica P.; Pogozheva, Irina D.; Jutkiewicz, Emily M.; Traynor, John R.; Mosberg, Henry I.

    2016-01-01

    In a previously described peptidomimetic series, we reported the development of bifunctional µ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: 1) probing bioavailability and improving metabolic stability, 2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the Κ-opioid receptor (KOR), and 3) improving in vivo efficacy. Here we establish that through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h. PMID:26524472

  9. Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

    PubMed

    Harland, Aubrie A; Yeomans, Larisa; Griggs, Nicholas W; Anand, Jessica P; Pogozheva, Irina D; Jutkiewicz, Emily M; Traynor, John R; Mosberg, Henry I

    2015-11-25

    In a previously described peptidomimetic series, we reported the development of bifunctional μ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h. PMID:26524472

  10. Synthesis, ligand-receptor modeling studies and pharmacological evaluation of novel 4-modified-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as potent and selective human A3 adenosine receptor antagonists.

    PubMed

    Colotta, Vittoria; Catarzi, Daniela; Varano, Flavia; Lenzi, Ombretta; Filacchioni, Guido; Martini, Claudia; Trincavelli, Letizia; Ciampi, Osele; Traini, Chiara; Pugliese, Anna Maria; Pedata, Felicita; Morizzo, Erika; Moro, Stefano

    2008-06-01

    The study of some 4-substituted-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, designed as hA(3) adenosine receptor antagonists, is reported. The new compounds bear on the four-position different acylamino, sulfonylamino, benzylureido and benzyloxy moieties, which have also been combined with a para-methoxy group on the 2-phenyl ring or with a nitro residue at the six-position. Many derivatives show high hA(3) adenosine receptor affinities and selectivities both versus hA(1) and hA(2A) receptors. The observed structure-affinity relationships of this class of antagonists have been exhaustively rationalized using the recently published ligand-based homology modeling (LBHM) approach. The selected 4-bismethanesulfonylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (13), which shows high hA(3) affinity (K(i)=5.5nM) and selectivity versus hA(1), hA(2A) (both selectivity ratios>1800) and hA(2B) (cAMP assay, IC(50)>10,000nM) receptors, was tested in an in vitro rat model of cerebral ischemia, proving to be effective in preventing the failure of synaptic activity, induced by oxygen and glucose deprivation in the hippocampus, and in delaying the occurrence of anoxic depolarization. PMID:18468446

  11. Affinity, potency and efficacy of tramadol and its metabolites at the cloned human mu-opioid receptor.

    PubMed

    Gillen, C; Haurand, M; Kobelt, D J; Wnendt, S

    2000-08-01

    The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect. PMID:10961373

  12. Mixed-model QSAR at the glucocorticoid receptor: predicting the binding mode and affinity of psychotropic drugs.

    PubMed

    Spreafico, Morena; Ernst, Beat; Lill, Markus A; Smiesko, Martin; Vedani, Angelo

    2009-01-01

    The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily that affects immune response, development, and metabolism in target tissues. Glucocorticoids are widely used to treat diverse pathophysiological conditions, but their clinical applicability is limited by side effects. A prediction of the binding affinity toward the GR would be beneficial for identifying glucocorticoid-mediated adverse effects triggered by drugs or chemicals. By identifying the binding mode to the GR using flexible docking (software Yeti) and quantifying the binding affinity through multidimensional QSAR (software Quasar), we validated a model family based on 110 compounds, representing four different chemical classes. The correlation with the experimental data (cross-validated r(2)=0.702; predictive r(2)=0.719) suggests that our approach is suited for predicting the binding affinity of related compounds toward the GR. After challenging the model by a series of scramble tests, a consensus approach (software Raptor), and a prediction set, it was incorporated into our VirtualToxLab and used to simulate and quantify the interaction of 24 psychotropic drugs with the GR. PMID:19009570

  13. Different subtypes of opioid receptors have different affinities for G-proteins.

    PubMed

    Polastron, J; Jauzac, P

    1994-05-01

    In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes. PMID:7920183

  14. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    PubMed

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. PMID:26795018

  15. Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists

    PubMed Central

    Iyer, Malliga R.; Cinar, Resat; Liu, Jie; Godlewski, Grzegorz; Szanda, Gergö; Puhl, Henry; Ikeda, Stephen R.; Deschamps, Jeffrey; Lee, Yong-Sok; Steinbach, Peter J.

    2015-01-01

    6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain-penetrant compound 14g [5-(4-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(1R,2R)-2-hydroxy-cyclohexyl]-3-pyridinecarboxamide] and its peripherally restricted analog 14h [5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2-methoxyethoxy)-3-pyridinecarboxamide], have been recently introduced as selective, high-affinity antagonists of the human cannabinoid-1 receptor (hCB1R). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14g and 14h. Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists and invalidates rodent models for assessing the antiobesity efficacy of 14g and 14h. PMID:26013543

  16. Toward Selective Drug Development for the Human 5-Hydroxytryptamine 1E Receptor: A Comparison of 5-Hydroxytryptamine 1E and 1F Receptor Structure-Affinity RelationshipsS⃞

    PubMed Central

    Klein, Michael T.; Dukat, Małgorzata; Glennon, Richard A.

    2011-01-01

    The 5-hydroxytryptamine (5-HT) 1E receptor is highly expressed in the human frontal cortex and hippocampus, and this distribution suggests the function of 5-HT1E receptors might be linked to memory. To test this hypothesis, behavioral experiments are needed. Because rats and mice lack a 5-HT1E receptor gene, knockout strategies cannot be used to elucidate this receptor's functions. Thus, selective pharmacological tools must be developed. The tryptamine-related agonist BRL54443 [5-hydroxy-3-(1-methylpiperidin-4-yl)-1H-indole] is one of the few agents that binds 5-HT1E receptors with high affinity and some selectively; unfortunately, it binds equally well to 5-HT1F receptors (Ki ≈ 1 nM). The differences between tryptamine binding requirements of these two receptor populations have never been extensively explored; this must be done to guide the design of analogs with greater selectivity for 5-HT1E receptors versus 5-HT1F receptors. Previously, we determined the receptor binding affinities of a large series of tryptamine analogs at the 5-HT1E receptor; we now examine the affinities of this same series of compounds at 5-HT1F receptors. The affinities of these compounds at 5-HT1E and 5-HT1F receptors were found to be highly correlated (r = 0.81). All high-affinity compounds were full agonists at both receptor populations. We identified 5-N-butyryloxy-N,N-dimethyltryptamine as a novel 5-HT1F receptor agonist with >60-fold selectivity versus 5-HT1E receptors. There is significant overlap between 5-HT1E and 5-HT1F receptor orthosteric binding properties; thus, identification of 5-HT1E-selective orthosteric ligands will be difficult. The insights generated from this study will inform future drug development and molecular modeling studies for both 5-HT1E and 5-HT1F receptors. PMID:21422162

  17. Similarities and differences in affinity and binding modes of tricyclic pyrimido- and pyrazinoxanthines at human and rat adenosine receptors.

    PubMed

    Szymańska, Ewa; Drabczyńska, Anna; Karcz, Tadeusz; Müller, Christa E; Köse, Meryem; Karolak-Wojciechowska, Janina; Fruziński, Andrzej; Schabikowski, Jakub; Doroz-Płonka, Agata; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna

    2016-09-15

    A new series of 32 pyrimido- and 5 tetrahydropyrazino[2,1-f]purinediones was obtained and evaluated for their adenosine receptors (ARs) affinities. The 1,3-dibutyl derivative of 9-(4-(2-(dimethylamino)ethoxy)phenyl)-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione was found to be the most potent A1 AR antagonist of the present series, showing selectivity over the other AR subtypes. The structure-activity for the obtained purinediones was established. Docking experiments of the investigated library to homology models of the human and rat A1 and A2A ARs allowed to compare the expected binding modes for selected compounds. The detailed analysis of binding cavities within individual AR subtypes indicated small but significant structural variations that may underlie the observed differences in binding affinities of purinediones at particular subtypes and species. PMID:27485602

  18. 2-Phenylpyrazolo[4,3-d]pyrimidin-7-one as a new scaffold to obtain potent and selective human A3 adenosine receptor antagonists: new insights into the receptor-antagonist recognition.

    PubMed

    Lenzi, Ombretta; Colotta, Vittoria; Catarzi, Daniela; Varano, Flavia; Poli, Daniela; Filacchioni, Guido; Varani, Katia; Vincenzi, Fabrizio; Borea, Pier Andrea; Paoletta, Silvia; Morizzo, Erika; Moro, Stefano

    2009-12-10

    A molecular simplification approach of previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones was applied to design 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives as new human A(3) adenosine receptor antagonists. Substituents with different lipophilicity and steric hindrance were introduced at the 5-position of the bicyclic scaffold (R(5) = H, Me, Et, Ph, CH(2)Ph) and on the 2-phenyl ring (OMe, Me). Most of the synthesized derivatives were highly potent hA(3) adenosine receptor antagonists, the best being the 2-(4-methoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-one (K(i) = 1.2 nM). The new compounds were also highly selective, being completely devoid of affinity toward hA(1), hA(2A), and hA(2B) adenosine receptors. On the basis of the recently published human A(2A) receptor crystallographic information, we propose a novel receptor-driven hypothesis to explain both A(3) AR affinity and A(3) versus A(2A) selectivity profiles of these new antagonists. PMID:19743865

  19. Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations

    PubMed Central

    Jones, RL; Woodward, DF; Wang, JW; Clark, RL

    2011-01-01

    BACKGROUND AND PURPOSE The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed. PMID:20973775

  20. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    SciTech Connect

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-12-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

  1. 5-HT1A receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

    PubMed Central

    Watson, J; Collin, L; Ho, M; Riley, G; Scott, C; Selkirk, J V; Price, G W

    2000-01-01

    It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT1A receptors. Saturation studies using [3H]-8-OH-DPAT and [3H]-MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5-HT1A receptors and rat cortex. In recombinant cells, [3H]-MPPF labelled 3–4 fold more sites than [3H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [3H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT1A receptors but did not bind to native tissue 5-HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5-HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. Receptor agonists inhibited [3H]-MPPF binding from recombinant 5-HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]-8-OH-DPAT and [3H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]-MPPF and [3H]-8-OH-DPAT in a monophasic manner. Functional evaluation of compounds, using [35S]-GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. [3H]-8-OH-DPAT : [3H]-MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]-8-OH-DPAT : [3H]-spiperone pKi difference with functional intrinsic activity (r=0.96). Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT1A receptors in which

  2. Temperature dependence of high-affinity CCK receptor binding and CCK internalization in rat pancreatic acini

    SciTech Connect

    Williams, J.A.; Bailey, A.C.; Roach, E. Univ. of California, San Francisco )

    1988-04-01

    {sup 125}I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperatures in isolated rat pancreatic acini. At 37{degree}C, acini readily bound and degraded {sup 125}I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (K{sub d}, 18 pM) and low-affinity (K{sub d}, 13 nM) binding sites. At 4{degree}C, acini bound only one-third as much {sup 125}I-CCK and degradation was essentially abolished. At 4{degree}C, CCK competition curves were consistent with a single class of low-affinity binding sites (K{sub d}, 19 nM). Internalization of {sup 125}I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound {sup 125}I-CCK, and this finding was validated for trypsin by electron microscope autotradiography. When internalization of {sup 125}I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.

  3. In Vivo Phenotypic Screening for Treating Chronic Neuropathic Pain: Modification of C2-Arylethynyl Group of Conformationally Constrained A3 Adenosine Receptor Agonists

    PubMed Central

    2015-01-01

    (N)-Methanocarba adenosine 5′-methyluronamides containing 2-arylethynyl groups were synthesized as A3 adenosine receptor (AR) agonists and screened in vivo (po) for reduction of neuropathic pain. A small N6-methyl group maintained binding affinity, with human > mouse A3AR and MW < 500 and other favorable physicochemical properties. Emax (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A3AR agonist, 2-(3,4-difluorophenylethynyl)-N6-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding Ki, hA3AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed particularly long in vivo duration (>3 h). Selected analogues were docked to an A3AR homology model to explore the environment of receptor-bound C2 and N6 groups. Various analogues bound with μM affinity at off-target biogenic amine (M2, 5HT2A, β3, 5HT2B, 5HT2C, and α2C) or other receptors. Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment. PMID:25422861

  4. Inhibition of Coxsackie B Virus Infection by Soluble Forms of Its Receptors: Binding Affinities, Altered Particle Formation, and Competition with Cellular Receptors

    PubMed Central

    Goodfellow, Ian G.; Evans, David J.; Blom, Anna M.; Kerrigan, Dave; Miners, J. Scott; Morgan, B. Paul; Spiller, O. Brad

    2005-01-01

    We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37°C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed. PMID:16140777

  5. Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains.

    SciTech Connect

    Zhou, H.; Sheng, S.; Compton, D.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.; Katzenellenbogen, B.; Nettles, K.; Greene, G.; Katzenellenbogen, J.; Biosciences Division; Univ. of Illinois; Univ. of Chicago; The Scripps Research Inst.

    2007-01-01

    2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

  6. Further characterization of the low and high affinity binding components of the thyrotropin receptor

    SciTech Connect

    McQuade, R.; Thomas, C.G. Jr.; Nayfeh, S.N.

    1986-05-29

    Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar /sup 125/I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited /sup 125/I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

  7. Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.

    PubMed

    Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C; Cogdill, Alexandria P; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M; June, Carl H; Zhao, Yangbing

    2015-09-01

    Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

  8. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice

    PubMed Central

    Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C.; Cogdill, Alexandria P.; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M.; June, Carl H.; Zhao, Yangbing

    2015-01-01

    Target-mediated toxicity is a major limitation in the development of chimeric antigen T cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues which express it at physiologic levels. A CAR-expressing T cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

  9. Synthesis, absolute configuration, conformational analysis and binding affinity properties of enantiomeric forms of DAU 5750, a novel M1-M3 muscarinic receptor antagonist.

    PubMed

    Turconi, M; Gozzo, A; Schiavi, G; Fronza, G; Mele, A; Bravo, P

    1994-12-01

    Both the enantiomeric forms of DAU 5750, a novel muscarinic receptor antagonist, have been synthesized in order to assess the relevance of configurational/conformational features for high affinity binding to muscarinic receptor subtypes. The attribution of absolute stereochemistry and conformational analysis by means of molecular modelling and NMR techniques are also reported. PMID:7788300

  10. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    SciTech Connect

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi ); Arai, Kenichi; Yokota, Takashi )

    1990-12-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of {sup 125}I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the {alpha} and {beta} subunits of the GM-CSF receptor, respectively.

  11. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  12. RELATIVE BINDING AFFINITY OF ENDOCRINE DISRUPTING CHEMICALS TO ESTROGEN RECEPTOR IN TWO SPECIES OF FRESHWATER FISH

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  13. Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking

    SciTech Connect

    Sheikh, S.P.; Williams, J.A. )

    1990-05-15

    Pharmacological studies indicate that peptide YY (PYY) and neuropeptide Y interact with multiple binding sites, categorized as Y1 and Y2 subtypes. In order to identify and structurally characterize the Y1 and Y2 receptors we covalently cross-linked (125I-Tyr36)PYY to its receptors. The Y2 receptor in rat hippocampus and rabbit kidney membranes was affinity labeled using different homo- and heterobifunctional cross-linking reagents. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography resulted in a major labeled protein band of Mr = 50,000 in both hippocampal and kidney membranes, which was unaffected by reducing agents. The Y1 receptor was analyzed in membranes from the MC-IXC human neuroblastoma cell line. Autoradiography revealed two labeled bands at Mr = 70,000 and 45,000. As the intensity of the Mr = 45,000 band was reduced by protease inhibitors, it is likely that this band is a degradation product of the larger band. Labeling of these proteins was obtained only when N-5-azido-2-nitrobenzoyloxysuccinimide was employed for cross-linking followed by exposure to UV light. Labeling of the two cross-linked bands was unaffected by reducing agents. The binding of radiolabeled PYY and the intensity of the cross-linked bands, for both the Y1 and Y2 receptors, were inhibited similarly in a dose-dependent manner by increasing concentrations of unlabeled PYY. When exposed to agarose-coupled lectins, the detergent-solubilized Y1 receptor-hormone complex was completely adsorbed by wheat germ agglutinin and partially by ricin communis II. The cross-linked Y2 receptor was almost totally adsorbed by wheat germ agglutinin-agarose and partially adsorbed by concanavalin A. The adsorptions were in all cases blocked by the appropriate hapten sugar.

  14. Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

    PubMed

    Pullinger, C R; Hennessy, L K; Chatterton, J E; Liu, W; Love, J A; Mendel, C M; Frost, P H; Malloy, M J; Schumaker, V N; Kane, J P

    1995-03-01

    Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose

  15. Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers.

    PubMed

    Ebert, Kristin; Wiemer, Jens; Caballero, Julio; Köckerling, Martin; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2015-09-01

    Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions. PMID:26189032

  16. Fluctuations in the affinity and concentration of insulin receptors on circulating monocytes of obese patients: effects of starvation, refeeding, and dieting.

    PubMed Central

    Bar, R S; Gorden, P; Roth, J; Kahn, C R; De Meyts, P

    1976-01-01

    The binding of 125I-insulin to insulin receptors on circulating monocytes of obese patients and normal volunteers has been determined under various dietary states. In the basal, fed state the monocytes of obese patients with clinical insulin resistance (n= 6) bound less insulin than normals (n =10) because of a decrease in insulin receptor concentration (obese = 6,000-13,000 sites per monocyte versus normals 15,000-28,000 sites per monocyte). The single obese patient without evidence of clinical insulin resistance demonstrated normal binding of insulin with 16,000 sites per monocyte. In all patients, the total receptor concentration was inversely related to the circulating levels of insulin measured at rest after an overnight fast. For the obese patients with basally depressed insulin binding, a 48-72-h fast lowered circulating insulin and increased binding to normal levels but only at low hormone concentrations; this limited normalization of 125I-insulin binding was associated with increased receptor affinity for insulin without change in receptor concentration. Refeeding after the acute fasting periods resulted in return to the elevated plasma insulin levels, the basal receptor affinity, and the depressed insulin binding observed in the basal, fed state. Chronic diet restored plasma insulin levels, insulin binding, and receptor concentration to normal without change in affinity. When the data from this study are coupled with previous in vivo and in vitro findings they suggest that the insulin receptor of human monocytes is more sensitive to regulation by ambient insulin than the receptors of obese mice and cultured human lymphocytes. The results further indicate than an insulin receptor undergoes in vivo modulation of its interaction with insulin by changing receptor concentration and by altering the affinity of existing receptors. Images PMID:993336

  17. Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.

    PubMed

    Yu, J; Xie, L Y; Abou-Samra, A B

    1996-01-01

    The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF. PMID:8536612

  18. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  19. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  20. Divergent activities of osteogenic BMP2, and tenogenic BMP12 and BMP13 independent of receptor binding affinities

    PubMed Central

    Berasi, Stephen P; Varadarajan, Usha; Archambault, Joanne; Cain, Michael; Souza, Tatyana A; Abouzeid, Abe; Li, Jian; Brown, Christopher T; Dorner, Andrew J; Seeherman, Howard J; Jelinsky, Scott A

    2011-01-01

    Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity. PMID:21702718

  1. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  2. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.

    PubMed

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the "AGIA" tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10-9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  3. Autoregulation by the Juxtamembrane Region of the Human Ephrin Receptor Tyrosine Kinase A3 (EphA3)

    SciTech Connect

    Davis, Tara L.; Walker, John R.; Loppnau, Peter; Butler-Cole, Christine; Allali-Hassani, Abdellah; Dhe-Paganon, Sirano

    2008-07-08

    Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamemebrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.

  4. Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.

    PubMed

    Lipp, Anna-Maria; Ji, Bozhi; Hager, Roland; Haas, Sandra; Schweiggl, Simone; Sonnleitner, Alois; Haselgrübler, Thomas

    2015-12-15

    Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged β3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment. PMID:26210593

  5. Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase.

    PubMed

    Neve, K A; Cox, B A; Henningsen, R A; Spanoyannis, A; Neve, R L

    1991-06-01

    An aspartate residue corresponding to aspartate-80 of dopamine D2 receptors is strictly conserved among receptors that couple to guanine nucleotide-binding proteins. Mutation of this residue alters the function of several classes of neurotransmitter receptors. Dopamine D2 receptors couple to the guanine nucleotide-binding protein Gi to inhibit adenylyl cyclase (ATP-pyrophosphate-lyase, cyclizing; EC 4.6.1.1). Like other Gi-coupled receptors, the binding of agonists and some antagonists to D2 receptors is sensitive to pH and sodium. In the present report, we demonstrate that substitution of an alanine or glutamate residue for aspartate-80 severely impairs inhibition of adenylyl cyclase by D2 receptors and also abolishes or decreases the regulation of the affinity of D2 receptors for agonists and substituted benzamide antagonists by sodium and pH. Our data support the hypothesis that the conformation of D2 receptors is maintained by interactions of monovalent cations with aspartate-80. The regulation of D2 receptors by this interaction has important consequences for the affinity of D2 receptors for ligands and for signal transduction by D2 receptors. PMID:1828858

  6. Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5

    PubMed Central

    Forse, Garry Jason; Uson, Maria Loressa; Nasertorabi, Fariborz; Kolatkar, Anand; Lamberto, Ilaria; Pasquale, Elena Bianca; Kuhn, Peter

    2015-01-01

    The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins. PMID:25993310

  7. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa. PMID:8183950

  8. PET studies with low and high affinity dopamine D2 receptor radioligands: Effects of 4-hydroxybutyrate (4HB)

    SciTech Connect

    Gatley, S.J.; Fowler, J.S.; Dewey, S.

    1994-05-01

    D2 radioligands of varying affinities have been developed as PET and SPECT radiotracers, but no consensus has been reached on the abilities of these tracers to quantify D2 receptor concentrations in vivo. Amongst other differences, competition of the radioligand with endogenous DA is expected to depend on affinity for the D2 receptor, so that changes in DA might confound estimates of Bmax. We examined the uptake and kinetics if C-11 raclopride (RAC; Kd = 1.2 nM) and C-11 N-methylspiperone (NMS); Kd = 75 pM in baboon striatum after pretreatment with 4HB (200 mg/Kg, i/v) which inhibits DA release by nigrostriatal nerve terminals. While 4HB diminished uptake (%ID/g) of NMS, it prolonged tissue retention of RAC, confirming previous observations in rodent models. Logan (for RAC) and Patlak (for NMS) plots gave changes of +24% and -20%, respectively, between control and 4HB treated animals. Since decreased competition with DA should increase uptake of NMS as well as RAC the paradoxical decrease in NMS uptake could be due to a second synaptic effect of DA, such as a decrease in agonist mediated internalization of NMS. Alternatively, it could result from an independent effect of 4HB, perhaps related to this drug`s ability to induce anesthesia and to depress cerebral glucose utilization. Although previous work in the rat suggests that 4HB does not alter brain blood flow, we found O-15 water that baboon striatal blood flow was decreased 22% and 42% at 30 and 60 minutes, respectively, after 4HB. Smaller changes were seen in cerebellar blood flow. Though a 4HB induced decrease in blood flow does not rule out a DA mediated alteration in D2 receptor Bmax or Kd for NMS, or other factor, it is unnecessary to invoke this to account for our results.

  9. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency

    PubMed Central

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M.; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G.; Marini, Pietro; Pertwee, Roger G.; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-01-01

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ9-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3–4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [3H]CP55,940 displacement and its effect on [35S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [35S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes. PMID:26124120

  10. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

    PubMed

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G; Marini, Pietro; Pertwee, Roger G; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-07-14

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes. PMID:26124120

  11. Differences in affinities between the homologous and the heterologous rabbit prolactin-receptor interaction with respect to proliferation and differentiation activities.

    PubMed

    Petridou, Barbara

    2015-03-01

    Interspecies differences in PRL-receptor binding and their relationship with bioactivity deserve investigation since cross-reactivity is relevant to the design of many experiments. We have previously shown that the lower affinity of rabbit prolactin (rbPRL) binding to its homologous receptor is due to its faster and more complete dissociation compared with that of ovine PRL (oPRL). In order to obtain sufficient amounts of rbPRL to study the functional consequences of its low affinity homologous interaction, rbPRL was expressed recombinantly in Escherichia coli (rec rbPRL) as insoluble inclusion bodies, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric rec rbPRL. Proper renaturation of rec rbPRL was evidenced by comparison of its CD spectra, binding parameters and bioactivity with those determined for the rbPRL. The binding potency of rec rbPRL to its receptor, expressed either endogenously in the mammary gland or recombinantly in mammalian cells is one log unit lower than that to the receptor expressed recombinantly in insect cells. This difference is probably related to differences in cell-dependent receptor densities. The proliferation potency of rbPRL or rec rbPRL was one log unit lower than that of oPRL, consistent with its lower binding affinity, but the differentiation potencies of these PRLs were similar. Thus, the proliferation activity is sensitive to PRL-receptor affinity and dissociation kinetics, whereas the differentiation response is marginally modulated. PMID:25449135

  12. Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

    PubMed Central

    Kitabatake, T.; Moaddel, R.; Cole, R.; Gandhari, M.; Frazier, C.; Hartenstein, J.; Rosenberg, A.; Bernier, M.; Wainer, I. W.

    2008-01-01

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  13. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

    PubMed

    Kitabatake, T; Moaddel, R; Cole, R; Gandhari, M; Frazier, C; Hartenstein, J; Rosenberg, A; Bernier, M; Wainer, I W

    2008-11-15

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  14. Discovery of the first small-molecule opioid pan antagonist with nanomolar affinity at mu, delta, kappa, and nociceptin opioid receptors.

    PubMed

    Zaveri, Nurulain T; Journigan, V Blair; Polgar, Willma E

    2015-04-15

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the

  15. Discovery of the First Small-Molecule Opioid Pan Antagonist with Nanomolar Affinity at Mu, Delta, Kappa, and Nociceptin Opioid Receptors

    PubMed Central

    2016-01-01

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [35S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure–activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [35S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3–6-fold more potent than those of JDTic. AT-076 is the

  16. 5-HT2 receptor affinity, docking studies and pharmacological evaluation of a series of 1,3-disubstituted thiourea derivatives.

    PubMed

    Bielenica, Anna; Kędzierska, Ewa; Koliński, Michał; Kmiecik, Sebastian; Koliński, Andrzej; Fiorino, Ferdinando; Severino, Beatrice; Magli, Elisa; Corvino, Angela; Rossi, Ilaria; Massarelli, Paola; Kozioł, Anna E; Sawczenko, Aleksandra; Struga, Marta

    2016-06-30

    A series of 10 thiourea derivatives have been synthesized by the reaction of aromatic amine with a substituted aryl (compounds 1-3, 6-8) and alkylphenyl (4, 5, 9, 10) isothiocyanates. Their in vitro and in vivo pharmacological properties were studied. Among the evaluated compounds, two displayed very high affinity for the 5-HT2A receptor (1-0.043 nM and 5-0.6 nM), being selective over the 5-HT2C receptor. Derivatives 3, 5, 9, 10 by 70-89% diminished L-5-HTP-induced head twitch episodes. Compounds 1 and 5 as the 5-HT2A receptor antagonists produced a dose-dependent decrease in the number of DOI-elicited HTR. Compounds 1-5 strongly reduced amphetamine-evoked hyperactivity in rodents. In another test, 1 and 2 caused hyperthermia in mice, whereas 9 and 10 led to hypothermia. Antinociceptive and anticonvulsant properties of selected derivatives were demonstrated. Molecular docking studies using a homology model of 5-HT2A revealed a significant role of hydrogen bonds between both thiourea NH groups and Asp155/Tyr370 residues, as well as π-π interaction with Phe339. PMID:27061981

  17. Changes in parathyroid hormone receptor binding affinity during egg laying: implications for calcium homeostasis in chicken.

    PubMed

    Yasuoka, T; Kawashima, M; Takahashi, T; Iwata, A; Oka, N; Tanaka, K

    1996-12-01

    Parathyroid hormone (PTH) receptor bindings were examined in the membrane fraction of the calvaria and the kidney of the hen by the use of [125I]PTH-related protein (PTHrP) binding assays. The binding specificity, reversibility, and saturation of the receptor were demonstrated. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were obtained by Scatchard analyses. In both calvaria and kidney, Kd and Bmax values decreased at 3 h before oviposition in egg-laying hens, but not in nonlaying hens. Administration of 17 beta-estradiol or progesterone in vivo caused a decrease in the Kd and Bmax values. Ionized calcium concentrations in the blood plasma showed a decrease at 13 h before oviposition. The results suggest that the PTH receptor binding in the calvaria and the kidney is affected by ovarian steroid hormones and may play a role in maintaining the calcium homeostasis in the egg-laying hen. PMID:8970893

  18. Does the tissue concentration in receptor binding studies change the affinity of the labelled ligand?

    PubMed

    Ensing, K; De Zeeuw, R A

    1984-12-14

    When the tissue concentration in a radioreceptor assay for anticholinergic drugs was varied in order to obtain optimum conditions, and the receptor concentration Cr and the equilibrium dissociation constant KD were determined by Scatchard analysis, the KD increased with increasing tissue concentrations. This phenomenon was considered as an artefact caused by non-specific binding of the labelled ligand to constituents of the receptor preparation which were not completely retained on the glass-fibre filters used for the separation of bound and free fraction of radio-labelled ligand. The increase in KD in these experiments could be described with a mathematical model of the binding experiments. PMID:6514542

  19. Aggregation Limits Surface Expression of Homomeric GluA3 Receptors.

    PubMed

    Coleman, Sarah K; Hou, Ying; Willibald, Marina; Semenov, Artur; Möykkynen, Tommi; Keinänen, Kari

    2016-04-15

    AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway. PMID:26912664

  20. Self-propelled affinity biosensors: Moving the receptor around the sample.

    PubMed

    Wang, J

    2016-02-15

    Self-propelled nanomotors offer considerable promise for developing novel biosensing protocols involving 'on-the-fly' recognition events. This article reviews recent advances in using catalytic nanomotors for bioaffinity sensing and for isolating target biomolecules and cells from complex biological samples. A variety of receptors, attached to self-propelled nanoscale motors, can thus move around the sample and, along with the generated microbubbles, lead to greatly enhanced fluid transport and accelerated recognition process. Such operation addresses the challenges imposed by the slow analyte transport in designing sensitive bioaffinity assays. The recognition element can be attached onto the motor surface or embedded in the motor material itself. Receptor-functionalized nanomotors based on different biomolecular interactions have thus been shown extremely useful for rapid target isolation from complex biological samples without preparatory and washing steps. Tubular microengine microtransporters, functionalized with antibody, ss-DNA, aptamer or lectin receptors, are particularly useful for direct detection and isolation of proteins, nucleic acids, proteins or cancer cells. Micromotors with 'built-in' recognition, exploiting the selective binding properties of the outer layer of such micronegines, can also be used. Greatly enhanced analyte-receptor interactions can also be achieved through the increased fluid transport associated with the movement of unmodified micromotors. The attractive features of the new motion-based bioaffinity sensing and separation protocols open up new opportunities for diverse biomedical, environmental and security applications. PMID:26074332

  1. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  2. Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

    SciTech Connect

    Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

    1986-05-01

    The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions and subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.

  3. Synthesis and receptor binding of N-substituted tropane derivatives. High-affinity ligands for the cocaine receptor

    SciTech Connect

    Milius, R.A.; Saha, J.K.; Madras, B.K.; Neumeyer, J.L. )

    1991-05-01

    The synthesis and pharmacological characterization of a series of N-substituted 3-(4-fluorophenyl)tropane derivatives is reported. The compounds displayed binding characteristics that paralleled those of cocaine, and several had substantially higher affinity at cocaine recognition sites. Conjugate addition of 4-fluorophenyl magnesium bromide to anhydroecgonine methyl ester gave 2 beta-(carbomethoxy)-3 beta-(4-fluorophenyl)tropane (4a, designated CFT, also known as WIN 35,428) after flash chromatography. N demethylation of 4a was effected by Zn/HOAc reduction of the corresponding 2,2,2-trichloroethyl carbamate to give 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane (5), which was alkylated with allyl bromide to afford the N-allyl analogue, 6. The N-propyl analogue, 7, was prepared by catalytic reduction (Pd/C) of 6. The most potent analogue, 4a, was tritiated at a specific activity of 81.3 Ci/mmol. ({sup 3}H)4a bound rapidly and reversibly to caudate putamen membranes; the two-component binding curve typical of cocaine analogues was observed. Equilibrium was achieved within 2 h and was stable for at least 4 h. High- and low-affinity Kd values observed for ({sup 3}H)4a (4.7 and 60 nM, respectively) were more than 4 times lower than those for ({sup 3}H)cocaine, and the density of binding sites (Bmax = 50 pmol/g, high, and 290 pmol/g, low) for the two drugs were comparable. Nonspecific binding of ({sup 3}H)4a was 5-10% of total binding.

  4. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure based modeling methods

    PubMed Central

    Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

    2016-01-01

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure-Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R2=0.55 and CCR=0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R2=0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. PMID:25058446

  5. Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

    PubMed Central

    Piirainen, Henni; Hellman, Maarit; Tossavainen, Helena; Permi, Perttu; Kursula, Petri; Jaakola, Veli-Pekka

    2015-01-01

    Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. PMID:25692595

  6. Association of EP2 receptor and SLC19A3 in regulating breast cancer metastasis

    PubMed Central

    Cheuk, Isabella W; Shin, Vivian Y; Siu, Man T; Tsang, Julia Y; Ho, John C; Chen, Jiawei; Tse, Gary M; Wang, Xian; Kwong, Ava

    2015-01-01

    Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer patients have higher metastatic rate than patients with other breast cancer subtypes. Distant metastasis is one of the causes leading to the high mortality rates. Cyclooxygenase-2 (COX2) is associated with breast cancer metastasis and the downstream prostaglandin E2 (PGE2) exerted its effect through EP receptors (EP1-EP4). However, the exact molecular events of EP receptors in breast cancer metastasis remain undefined. Expressions of EP receptors were determined during cancer development in NOD-SCID mice inoculated with MB-231 and MB-231-EP2 clone. EP2 overexpressing stable clone was constructed to investigate the proliferation and invasion potentials in vivo and in vitro. Drug transporter array was used to identify EP2 receptor-associated drug transported genes in breast cancer metastasis. Localization of EP2 receptor in primary tissues and xenografts were examined by immunostaining. Stable EP2-expression cells formed larger tumors than parental cells in mice model and was highly expressed in both primary and metastatic tissues. Silencing of EP2 receptor by siRNA and antagonist (AH 6809) significantly decreased cell proliferation and invasion, concomitant with reduced MMP-2 and MMP-9 expressions. Results from array data showed that expression of SLC19A3 was markedly increased in EP2 siRNA transfected cells. Ectopic expression of SLC19A3 retarded cell proliferation, invasion and MMPs expressions. Notably, SLC19A3 had a lower expression in primary tissues and was negatively correlated with EP2 receptor expression. Our novel finding revealed that EP2 receptor regulated metastasis through downregulation of SLC19A3. Thus, targeting EP2-SLC19A3 signaling is a potential therapeutic therapy for treating metastatic breast cancer. PMID:26807319

  7. Association of EP2 receptor and SLC19A3 in regulating breast cancer metastasis.

    PubMed

    Cheuk, Isabella W; Shin, Vivian Y; Siu, Man T; Tsang, Julia Y; Ho, John C; Chen, Jiawei; Tse, Gary M; Wang, Xian; Kwong, Ava

    2015-01-01

    Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer patients have higher metastatic rate than patients with other breast cancer subtypes. Distant metastasis is one of the causes leading to the high mortality rates. Cyclooxygenase-2 (COX2) is associated with breast cancer metastasis and the downstream prostaglandin E2 (PGE2) exerted its effect through EP receptors (EP1-EP4). However, the exact molecular events of EP receptors in breast cancer metastasis remain undefined. Expressions of EP receptors were determined during cancer development in NOD-SCID mice inoculated with MB-231 and MB-231-EP2 clone. EP2 overexpressing stable clone was constructed to investigate the proliferation and invasion potentials in vivo and in vitro. Drug transporter array was used to identify EP2 receptor-associated drug transported genes in breast cancer metastasis. Localization of EP2 receptor in primary tissues and xenografts were examined by immunostaining. Stable EP2-expression cells formed larger tumors than parental cells in mice model and was highly expressed in both primary and metastatic tissues. Silencing of EP2 receptor by siRNA and antagonist (AH 6809) significantly decreased cell proliferation and invasion, concomitant with reduced MMP-2 and MMP-9 expressions. Results from array data showed that expression of SLC19A3 was markedly increased in EP2 siRNA transfected cells. Ectopic expression of SLC19A3 retarded cell proliferation, invasion and MMPs expressions. Notably, SLC19A3 had a lower expression in primary tissues and was negatively correlated with EP2 receptor expression. Our novel finding revealed that EP2 receptor regulated metastasis through downregulation of SLC19A3. Thus, targeting EP2-SLC19A3 signaling is a potential therapeutic therapy for treating metastatic breast cancer. PMID:26807319

  8. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

    SciTech Connect

    Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

    2014-10-01

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables

  9. Structure-based approach for the study of thyroid hormone receptor binding affinity and subtype selectivity.

    PubMed

    Wang, Fang-Fang; Yang, Wei; Shi, Yong-Hui; Cheng, Xiang-Rong; Le, Guo-Wei

    2016-10-01

    Thyroid hormone (TH) possesses the ability to lower cholesterol and improve cardiac performance, which have prompted the efforts to design analogs that can utilize the cholesterol-lowering property without adversely affecting heart function. In order to gain insights into the interaction mechanism for agonists at the active site of thyroid hormone receptor β (TRβ), quantitative structure-activity relationship (QSAR) models have been developed on TRβ agonists, significant statistical coefficients were obtained (CoMFA, R(2)cv, .732), (CoMSIA, R(2)cv, .853), indicating the internal consistency of the models, the obtained models were further validated using the test set, the acquired R(2)pred values .7054 and .7129 were in good agreement with the experimental results. The key amino acids affecting ligand binding were identified by molecular docking, and the detailed binding modes of the compounds with different activities were also determined. Furthermore, molecular dynamics (MD) simulations were conducted to assess the reliability of the derived models and the docking results. Moreover, TH exerts significant physiological effects through modulation of the two human thyroid hormone receptor subtypes. Because TRβ and TRα locate in different target cells, selective TR ligands would target specific tissues regulated by one receptor without affecting the other. Thus, the 3D information was analyzed to reveal the most relevant structural features involved in selectivity. The findings serve as the basis for further investigation into selective TRβ/TRα agonists. PMID:26510472

  10. High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

    PubMed

    Torres, C; Perlin, M H; Baquero, F; Lerner, D L; Lerner, S A

    2000-08-01

    This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed. PMID:10929874

  11. 3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

    PubMed

    Werle, E; Lenz, T; Strobel, G; Weicker, H

    1988-07-01

    The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off PMID:2853303

  12. Differential effects of chronic hyperammonemia on modulation of the glutamate-nitric oxide-cGMP pathway by metabotropic glutamate receptor 5 and low and high affinity AMPA receptors in cerebellum in vivo.

    PubMed

    Cabrera-Pastor, Andrea; Llansola, Marta; Reznikov, Vitaliy; Boix, Jordi; Felipo, Vicente

    2012-07-01

    Previous studies show that chronic hyperammonemia impairs learning ability of rats by impairing the glutamate-nitric oxide (NO)-cyclic guanosine mono-phosphate (cGMP) pathway in cerebellum. Three types of glutamate receptors cooperate in modulating the NO-cGMP pathway: metabotropic glutamate receptor 5 (mGluR5), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors. The aim of this work was to assess whether hyperammonemia alters the modulation of this pathway by mGluR5 and AMPA receptors in cerebellum in vivo. The results support that in control rats: (1) low AMPA concentrations (0.1mM) activate nearly completely Ca(2+)-permeable (glutamate receptor subunit 2 (GluR2)-lacking) AMPA receptors and the NO-cGMP pathway; (2) higher AMPA concentrations (0.3 mM) also activate Ca(2+)-impermeable (GluR2-containing) AMPA receptors, leading to activation of NMDA receptors and of NO-cGMP pathway. Moreover, the data support that chronic hyperammonemia: (1) reduces glutamate release and activation of the glutamate-NO-cGMP pathway by activation of mGluR5; (2) strongly reduces the direct activation by AMPA receptors of the NO-cGMP pathway, likely due to reduced entry of Ca(2+) through GluR2-lacking, high affinity AMPA receptors; (3) strongly increases the indirect activation of the NO-cGMP pathway by high affinity AMPA receptors, likely due to increased entry of Na(+) through GluR2-lacking AMPA receptors and NMDA receptors activation; (4) reduces the indirect activation of the NO-cGMP pathway by low affinity AMPA receptors, likely due to reduced activation of NMDA receptors. PMID:22521775

  13. Affinities and densities of high-affinity (/sup 3/H)muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    SciTech Connect

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-09-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using (/sup 3/H)muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using (/sup 3/H)flunitrazepam and (/sup 3/H)Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of (/sup 3/H)muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy.

  14. New analogues of epiboxidine incorporating the 4,5-dihydroisoxazole nucleus: synthesis, binding affinity at neuronal nicotinic acetylcholine receptors, and molecular modeling investigations.

    PubMed

    Dallanoce, Clelia; Magrone, Pietro; Bazza, Paola; Grazioso, Giovanni; Rizzi, Luca; Riganti, Loredana; Gotti, Cecilia; Clementi, Francesco; Frydenvang, Karla; De Amici, Marco

    2009-02-01

    A group of novel 4,5-dihydro-3-methylisoxazolyl derivatives, structurally related to epiboxidine (=(1R,4S,6S)-6-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]heptane), was prepared via 1,3-dipolar cycloaddition of acetonitrile oxide to different olefins. Target compounds 1a and 1b, 2a and 2b, 3, 4, and 5 were tested for affinity at neuronal nicotinic heteromeric (alpha4beta2) and homomeric (alpha7) acetylcholine receptors. Notably, diastereoisomers 1a and 1b were characterized by a massive drop of the affinity at the alpha4beta2 subtypes (K(i) values spanning the range 4.3-126 microM), when compared with that of epiboxidine (K(i)=0.6 nM). Therefore, the replacement of the 3-methylisoxazole ring of epiboxidine with the 4,5-dihydro-3-methylisoxazole nucleus is detrimental for the affinity at alpha4beta2 receptors. A comparable lack of affinity/selectivity for the two nAChR subtypes under study was evidenced for the remaining epiboxidine-related dihydroisoxazole derivatives 2a and 2b, and 3-5. Diastereoisomers 1a and 1b, and spirocyclic derivative 3 were docked into molecular models of the receptor subtypes under study, and their binding mode was compared with that of reference ligands endowed with high binding affinity. PMID:19235154

  15. Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors.

    PubMed

    Carlin, Jesse Lea; Tosh, Dilip K; Xiao, Cuiying; Piñol, Ramón A; Chen, Zhoumou; Salvemini, Daniela; Gavrilova, Oksana; Jacobson, Kenneth A; Reitman, Marc L

    2016-02-01

    Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist-induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non-brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia. PMID:26606937

  16. Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors

    PubMed Central

    Carlin, Jesse Lea; Tosh, Dilip K.; Xiao, Cuiying; Piñol, Ramón A.; Chen, Zhoumou; Salvemini, Daniela; Gavrilova, Oksana; Jacobson, Kenneth A.

    2016-01-01

    Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist–induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non–brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia. PMID:26606937

  17. Age-Related Yield of Adipose-Derived Stem Cells Bearing the Low-Affinity Nerve Growth Factor Receptor

    PubMed Central

    González-Garza, Maria Teresa; Cardenas-Lopez, Alejandro; Chavez-Castilla, Luis; Cruz-Vega, Delia Elva; Moreno-Cuevas, Jorge E.

    2013-01-01

    Adipose-derived stem cells (ADSCs) are a heterogeneous cell population that may be enriched by positive selection with antibodies against the low-affinity nerve growth factor receptor (LNGFR or CD271), yielding a selective cell universe with higher proliferation and differentiation potential. This paper addresses the need for determining the quantity of ADSCs positive for the CD271 receptor and its correlation with donor's age. Mononuclear cells were harvested from the lower backs of 35 female donors and purified using magnetic beads. Multipotency capacity was tested by the expression of stemness genes and through differentiation into preosteoblasts and adipocytes. A significant statistical difference was found in CD271+ concentrations between defined age intervals. The highest yield was found within women on the 30–40-year-old age range. CD271+ ADSCs from all age groups showed differentiation capabilities as well as expression of typical multipotent stem cell genes. Our data suggest that the amount of CD271+ cells correlates inversely with age. However, the ability to obtain these cells was maintained through all age ranges with a yield higher than what has been reported from bone marrow. Our findings propose CD271+ ADSCs as the primary choice for tissue regeneration and autologous stem cell therapies in older subjects. PMID:24376462

  18. NMR and computational evidence that high-affinity bradykinin receptor antagonists adopt C-terminal beta-turns.

    PubMed

    Kyle, D J; Blake, P R; Smithwick, D; Green, L M; Martin, J A; Sinsko, J A; Summers, M F

    1993-05-14

    Three tetrapeptides were prepared, each corresponding to the four C-terminal amino acid residues of highly potent, second-generation bradykinin receptor antagonists. The tetrapeptides are (IA) Ser-D-Phe-Oic-Arg, (IIA) Ser-D-Tic-Oic-Arg, and (IIIA) Ser-D-Hype(trans-propyl)-Oic-Arg. Solution conformations for each were determined by incorporating interproton distance restraints, determined by 2D NMR experiments performed in water at neutral pH, into a series of distance geometry/simulated annealing model building calculations. Similarly, systematic conformational analyses were performed for each using molecular mechanics calculations. Both the NMR-derived structures, as well as the calculated structures, are shown to adopt a beta-turn as the primary conformation. Excellent agreement between the predicted structures and the NMR-derived structures is demonstrated. Aside from being the first examples of linear tetrapeptides reported to be ordered in aqueous solvent, the results presented support the hypothesis that high-affinity bradykinin receptor antagonists must adopt C-terminal beta-turn conformations. PMID:8388469

  19. The Analysis of the Human High Affinity IgE Receptor FceRIa from Multiple Crystal Forms

    SciTech Connect

    Garman, S.C.; Sechi, S.; Kinet, J.-P.; Jardetzky, T.S.

    2010-03-05

    We have solved the structure of the human high affinity IgE receptor, Fc{var_epsilon}RI{alpha}, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc{var_epsilon}RI{alpha} with its natural ligand and thus to prevent a primary step in the allergic response.

  20. Mixed-model QSAR at the human mineralocorticoid receptor: predicting binding mode and affinity of anabolic steroids.

    PubMed

    Peristera, Ourania; Spreafico, Morena; Smiesko, Martin; Ernst, Beat; Vedani, Angelo

    2009-09-28

    We present a computational study on the human mineralocorticoid receptor (hMR) that is based on multi-dimensional quantitative structure-activity relationships (mQSAR). Therein, we identified the binding mode of 48 steroid and non-steroid homologues by flexible docking to the crystal structure (software Yeti) and quantified it using 6D-QSAR (software Quasar). The receptor surrogate, evolved using a genetic algorithm, converged at a cross-validated r2 of 0.810, and yielded a predictive r2 of 0.661. The model was challenged by a series of scramble tests and by consensus scoring (software Raptor: r2=0.844, predictive r(2)=0.620). The model was then employed to predict the binding affinity of 26 anabolic steroids, demonstrating to which extent they might disrupt the endocrine system via binding to the hMR. The model for the hMR was added to the VirtualToxLab, a technology developed by the Biographics Laboratory 3R, allows the identification of the endocrine-disrupting potential of drugs, chemicals and natural products in silico. PMID:19523507

  1. New 2-arylpyrazolo[3,4-c]quinoline derivatives as potent and selective human A3 adenosine receptor antagonists. Synthesis, pharmacological evaluation, and ligand-receptor modeling studies.

    PubMed

    Colotta, Vittoria; Catarzi, Daniela; Varano, Flavia; Capelli, Francesca; Lenzi, Ombretta; Filacchioni, Guido; Martini, Claudia; Trincavelli, Letizia; Ciampi, Osele; Pugliese, Anna Maria; Pedata, Felicita; Schiesaro, Andrea; Morizzo, Erika; Moro, Stefano

    2007-08-23

    This paper reports the study of some 2-arylpyrazolo[3,4-c]quinolin-4-ones, 4-amines, and 4-amino-substituted derivatives designed as human A3 adenosine receptor (AR) antagonists. Most of the herein reported compounds showed a nanomolar affinity toward the hA3 receptor subtype and different degrees of selectivity that resulted to be strictly dependent on the presence and nature of the substituent on the 4-amino group. Bulky and lipophilic acyl groups, as well as the benzylcarbamoyl residue, afforded highly potent and selective hA3 receptor antagonists. The selected 4-diphenylacetylamino-2-phenylpyrazoloquinoline (25) and 4-dibenzoylamino-2-(4-methoxyphenyl)pyrazoloquinoline (36), tested in an in vitro rat model of cerebral ischemia, prevented the irreversible failure of synaptic activity induced by oxygen and glucose deprivation in the hippocampus. The observed structure-affinity relationships of this class of antagonists were also exhaustively rationalized using the recently published ligand-based homology modeling (LBHM) approach. PMID:17665891

  2. PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

    PubMed Central

    Noberini, Roberta; Mitra, Sayantan; Salvucci, Ombretta; Valencia, Fatima; Duggineni, Srinivas; Prigozhina, Natalie; Wei, Ke; Tosato, Giovanna; Huang, Ziwei; Pasquale, Elena B.

    2011-01-01

    The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore

  3. 4-amido-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones as new potent and selective human A3 adenosine receptor antagonists. synthesis, pharmacological evaluation, and ligand-receptor modeling studies.

    PubMed

    Lenzi, Ombretta; Colotta, Vittoria; Catarzi, Daniela; Varano, Flavia; Filacchioni, Guido; Martini, Claudia; Trincavelli, Letizia; Ciampi, Osele; Varani, Katia; Marighetti, Federico; Morizzo, Erika; Moro, Stefano

    2006-06-29

    A structural investigation on some 4-amido-2-phenyl-1,2-dihydro-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, designed as human A3 adenosine receptor (hA3 AR) antagonists, is described. In the new derivatives, some acyl residues with different steric bulk were introduced on the 4-amino group, and their combination with the 4-methoxy group on the 2-phenyl moiety, and/or the 6-nitro/6-amino substituent on the fused benzo ring, was also evaluated. Most of the new derivatives were potent and selective hA3 AR antagonists. SAR analysis showed that hindering and lipophilic acyl moieties not only are well tolerated but even ameliorate the hA3 affinity. Interestingly, the 4-methoxy substituent on the appended 2-phenyl moiety, as well as the 6-amino group, always exerted a positive effect, shifting the affinity toward the hA3 receptor subtype. In contrast, the 6-nitro substituent exerted a variable effect. An intensive molecular modeling investigation was performed to rationalize the experimental SAR findings. PMID:16789747

  4. The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

    PubMed Central

    Roth, Bryan L.; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects. PMID:23527166

  5. A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

    PubMed

    Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

    2015-06-01

    Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. PMID:25969169

  6. Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide

    SciTech Connect

    Haring, R.; Kloog, Y.; Harshak-Felixbrodt, N.A.; Sokolovsky, M.

    1987-01-30

    Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for (/sup 3/H)TCP, (/sup 3/H)N-(1-(2-thienyl)cyclohexyl)piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for (/sup 3/H)TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K or Na). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by (/sup 3/H)azido phencyclidine is selectively inhibited by monovalent ions.

  7. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  8. Potential Modes of Interaction of 9-Aminomethyl-9,10-dihydroanthracene (AMDA) Derivatives with the 5-HT2A Receptor: A Ligand Structure-Affinity Relationship, Receptor Mutagenesis and Receptor Modeling Investigation⊕

    PubMed Central

    Runyon, Scott P.; Mosier, Philip D.; Roth, Bryan L.; Glennon, Richard A.; Westkaemper, Richard B.

    2011-01-01

    The effects of 3-position substitution of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on 5-HT2A receptor affinity were determined and compared to a parallel series of DOB-like 1-(2,5-dimethoxyphenyl)-2-aminopropanes substituted at the 4-position. The results were interpreted within the context of 5-HT2A receptor models that suggest that members of the DOB-like series can bind to the receptor in two distinct modes that correlate with the compounds’ functional activity. Automated ligand docking and molecular dynamics suggest that all of the AMDA derivatives, the parent of which is a 5-HT2A antagonist, bind in a fashion analogous to that for the sterically demanding antagonist DOB-like compounds. The failure of the F3406.52L mutation to adversely affect the affinity of AMDA and the 3-bromo derivative is consistent with the proposed modes of orientation. Evaluation of ligand-receptor complex models suggest that a valine/threonine exchange between the 5-HT2A and D2 receptors may be the origin of selectivity for AMDA and two substituted derivatives. PMID:18847250

  9. Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

    PubMed Central

    Chen, Sylvia; Webber, Matthew J.; Vilardaga, Jean-Pierre; Khatri, Ashok; Brown, Dennis; Ausiello, Dennis A.; Lin, Herbert Y.

    2011-01-01

    The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism. PMID:21828182

  10. [125I]AT-1012, a New High Affinity Radioligand for the α3β4 Nicotinic Acetylcholine Receptors

    PubMed Central

    Wu, Jinhua; Perry, David C.; Bupp, James E.; Jiang, Faming; Polgar, Willma E.; Toll, Lawrence; Zaveri, Nurulain T.

    2013-01-01

    Recent genetic and pharmacological studies have implicated the α3, β4 and a5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4- and α4β2 nAChR transfected into HEK cells as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at human α3β4α5 nAChR in a human α3β4aα nAChR transfected cell line. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells, and [3H]epibatidine binding studies showed that AT-1012 had over 100-fold binding selectivity for the α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label the α3β4 nAChR in rat brain slices in vitro using autoradiography which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We

  11. Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

    PubMed Central

    2015-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands. PMID:25133934

  12. Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor.

    PubMed

    Kamio, M; Uchiyama, T; Arima, N; Itoh, K; Ishikawa, T; Hori, T; Uchino, H

    1990-01-01

    Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta

  13. The receptor binding affinity of monocyclic [Ala3,Xaa11]endothelin-1 analogs correlates with inducible helix length.

    PubMed

    Andersen, N H; Harris, S M; Lee, V G; Liu, E C; Moreland, S; Hunt, J T

    1995-02-01

    Endothelin-1, a bicyclic 21-amino acid peptide with disulfide bridges between cysteines 1 and 15 as well as between cysteines 3 and 11, has been reported to be partially helical based on both CD and NMR data. However, this remains an area of controversy with some claims that CD data indicate no alpha-helical structure (Calas, B.; Harricane, M.-C.; Gulmard, L.; Heitz, F.; Mendre, C.; Chabrier, P.E.; Bennes, R. Peptide Res. 1992, 5, 97) and a recent X-ray crystal structure placing the helix at a different locus (Janes, R.W.; Peapus, D.H.; Wallace, B.A. Structural Biology 1994, 1, 311). The CD studies reported herein indicate that the helical structures reported in NMR studies (e.g. Andersen, N.H.; Chen, C.; Marschner, T.M.; Krystek, Jr. S.R.; Bassolino, D.A. Biochemistry 1992, 31, 1280) apply to pure aqueous media as well. The helix located from Lys9 to the Cys15/His16 juncture is ca 75% populated in pH 4 aqueous buffer. Titration difference CDs reveal that the helix extent increases by one to two residues and that the 'helical conformation' is more completely populated upon addition of TFE to 50+ volume-%. Comparison with a more helical analog suggests that the helix propagates towards (but not to the end of) the C-terminus upon fluoroalcohol addition. A variety of monocyclic derivatives of [Nle7] ET-1 lacking the 3,11-disulfide were evaluated for biological activity and examined by TFE titration difference CD. The series included an Aib11 and a Pro11 analog. The helix promoting Aib analog was the most active while the Pro analog exhibited significantly lower vasoconstrictor activity and binding affinity for the ETA receptor. All of the monocyclic analogs became significantly more helical upon addition of fluoroalcohols. The inclusion of a proline residue at position 11 does not preclude helix formation upon addition of fluoroalcohols. Rather, helix formation is relatively easily induced but limited to a 5 residue span. Apparently this is insufficient to orient

  14. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    SciTech Connect

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-02-15

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (Fc{epsilon}RI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with Fc{epsilon}RI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.

  15. The Synthesis of 1,3,5-triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter.

    PubMed

    Latacz, Gniewomir; Kechagioglou, Petros; Papi, Rigini; Łażewska, Dorota; Więcek, Małgorzata; Kamińska, Katarzyna; Wencel, Przemysław; Karcz, Tadeusz; Schwed, Johannes S; Stark, Holger; Kyriakidis, Dimitrios A; Kieć-Kononowicz, Katarzyna

    2016-08-01

    The involvement of histamine and H4 receptor (H4 R) in cancer has been investigated recently using the H4 R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H4 receptor (H4 R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H4 R antagonists JNJ7777120 structure or 1,3,5-triazine scaffold were synthesized, evaluated for histamine H4 R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H4 R with Ki value >100 μm. These compounds showed no significant effect on neuroblastoma IMR-32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6, another JNJ7777120 analogue, showed the highest effect on IMR-32 viability with calculated IC50 = 23.27 μm. The 1,3,5-triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5-triazine derivative 11 with the para-bromo substituent showed the highest affinity against H4 R with Ki value of 520 nm and may be considered as a new lead structure. PMID:26931395

  16. Behavioral interactions between ethanol and imidazodiazepines with high affinities for benzodiazepine receptors

    SciTech Connect

    Lister, R.G.

    1988-01-01

    The intrinsic effect of two imidazodiazepines RO 15-3505 and RO 17-1812 on the behavior of mice in a holeboard test were investigated. The interactions of these two drugs with ethanol were also studied. RO 15-3505 failed to significantly alter either exploratory head-dipping or locomotor activity when administered alone but doses of 0.75 and 1.5 mg/kg reversed the reduction in the number of head-dips caused by ethanol and partially reversed ethanol's locomotor stimulant action. In contrast, RO 17-1812 increased locomotor activity when administered alone, and enhanced the reduction in exploration caused by ethanol. Neither RO 15-3505 nor RO 17-1812 altered blood alcohol concentrations suggesting a pharmacodynamic basis for these interactions. The results suggest that in the holeboard test the interactions of imidazodiazepines with ethanol are related to the nature of their interaction with benzodiazepine receptors, inverse agonists antagonising and agonists enhancing ethanol's effects on exploration.

  17. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    PubMed Central

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  18. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

    PubMed

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  19. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  20. Nicotine ameliorates NMDA receptor antagonist-induced deficits in contextual fear conditioning through high-affinity nicotinic acetylcholine receptors in the hippocampus.

    PubMed

    André, Jessica M; Leach, Prescott T; Gould, Thomas J

    2011-03-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV)-induced disruption of contextual fear conditioning in male C57BL/6J mice, using direct drug infusion and selective nAChR antagonists to define the brain regions and the nAChR subtypes involved. Mice treated with MK-801 showed a deficit in contextual fear conditioning that was ameliorated by nicotine. Direct drug infusion demonstrated that the NMDAR antagonists disrupted hippocampal function and that nicotine acted in the dorsal hippocampus to ameliorate the deficit in learning. The high-affinity nAChR antagonist Dihydro-β-erythroidine hydrobromide (DhβE) blocked the effects of nicotine on MK-801-induced deficits while the α7 nAChR antagonist methyllycaconitine citrate salt hydrate (MLA) did not. These results suggest that NMDARs and nAChRs may mediate similar hippocampal processes involved in contextual fear conditioning. Furthermore, these results may have implications for developing effective therapeutics for the cognitive deficits associated with schizophrenia because a large subset of patients with schizophrenia exhibit cognitive deficits that may be related to NMDAR dysfunction and smoke at much higher rates than the healthy population, which may be an attempt to ameliorate cognitive deficits. PMID:21167848

  1. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    SciTech Connect

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  2. Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites.

    PubMed Central

    Hennig, B; Koehler, H; Neupert, W

    1983-01-01

    Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria. PMID:6308663

  3. Triaryl-Substituted Schiff Bases Are High-Affinity Subtype-Selective Ligands for the Estrogen Receptor

    PubMed Central

    2015-01-01

    We have explored the isoelectronic replacement of the C=C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C=N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C=N for C=C substitution might be more widely considered as a general strategy for preparing drug analogues. PMID:24708493

  4. (3H) 5,7-dichlorokynurenic acid, a high affinity ligand for the NMDA receptor glycine regulatory site

    SciTech Connect

    Hurt, S.D.; Baron, B.M. )

    1991-01-01

    The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. The authors have prepared (3H) 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield (3H) 5,7-DCKA with a specific activity of 17.6 Ci/mmol. (3H) 5,7-DCKA bound to rat brain synaptosomes with a Kd of 69 {plus minus} 23 nM and Bmax = 14.5 {plus minus} 3.2 pmoles/mg protein. Binding was 65-70% specific at 10 nM (3H) 5,7-DCKA. This ligand is thus more selective and has higher affinity than (3H) glycine, in addition to being an antagonist.

  5. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

    PubMed

    Wang, Yu-Chuan; Chin, Ko-Hsin; Tu, Zhi-Le; He, Jin; Jones, Christopher J; Sanchez, David Zamorano; Yildiz, Fitnat H; Galperin, Michael Y; Chou, Shan-Ho

    2016-01-01

    C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date. PMID:27578558

  6. Sodium-dependent isomerization of dopamine D-2 receptors characterized using [125I]epidepride, a high-affinity substituted benzamide ligand.

    PubMed

    Neve, K A; Henningsen, R A; Kinzie, J M; De Paulis, T; Schmidt, D E; Kessler, R M; Janowsky, A

    1990-03-01

    We have characterized the in vitro binding of a new ligand, [125I]epidepride, and used this substituted benzamide to assess the sensitivity of dopamine D-2 receptors to sodium. Both direct and indirect binding studies with [125I]epidepride and unlabeled epidepride, respectively, demonstrated that the affinity of D-2 receptors for the ligand was decreased from 20 to 30 pM in the presence of sodium to 350 to 500 pM in the absence of sodium. The density of binding sites for [125I]epidepride was identical in the presence and absence of NaCl. The time courses for association of [125I]epidepride to and dissociation from D-2 receptors in the presence of sodium were not consistent with simple bimolecular reactions, suggesting the possibility of a sodium-dependent ligand-induced receptor isomerization. Thus, dissociation of [125I]epidepride was biphasic in the presence of sodium, but monophasic in the absence of sodium. The rank order of potency for inhibition of [125I]epidepride binding by drugs was identical in rat striatum and cells expressing a D-2 receptor cDNA, and similar to the previously described pharmacological profile of D-2 receptors labeled by [3H]spiperone. [125I]Epidepride bound to two classes of binding sites in rat medial prefrontal cortex. One class, present at a density of 10 fmol/mg of protein and with a Kd value of approximately 40 pM, was pharmacologically indistinguishable from D-2 receptors in striatum and transfected cells. The pharmacological profile of the second class of sites was similar to that of alpha-2 adrenergic receptors. [125I]Epidepride had 50- to 100-fold lower affinity (approximately 2 nM) for alpha-2 receptors than for D-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2138666

  7. Expression of low-, intermediate-, and high-affinity IL-2 receptors on B cell lines derived from patients with undifferentiated lymphoma of Burkitt's and non-Burkitt's types

    SciTech Connect

    Benjamin, D.; Rosolen, A.; Wormsley, S.B.; DeBault, L.E.; Colamonici, O.R. )

    1990-08-01

    IL-2 receptors on T cells exist in at least three forms which differ in their ligand-binding affinity. The low-affinity IL-2 receptor (IL-2R) consists of the 55-kDa Tac protein (p55 alpha), the intermediate-affinity site corresponds to the 70-kDa molecule (p70 beta), and the high-affinity IL-2R consists of a noncovalent heterodimeric structure involving both p55 alpha and p70 beta. We studied 24 B cell lines (8 EBV-negative and 16 EBV-positive) for IL-2R expression in the presence or absence of the tumor promoter, teleocidin. 125I-IL-2 radioreceptor binding assays and crosslinking studies demonstrated the sole expression of p55 alpha in EBV-negative cell lines only, whereas p55 alpha present in EBV-positive cell lines was always associated with p70 beta to construct high-affinity IL-2R. p70 beta was not detected in any of the EBV-negative cell lines, but was expressed on most of the EBV-positive cell lines (13 of 16). Our data also indicate that the expression of p55 alpha and p70 beta by radiolabeling correlates with their expression in flow cytometry, and that a large excess of p55 alpha is required to construct high-affinity IL-2R. Coexpression of p55 alpha and p70 beta on human B cells contributed to constructing high-affinity IL-2R hybrid complex as shown by rapid association rate contributed by p55 alpha and slow dissociation rate by p70 beta; teleocidin's ability to induce p55 alpha on cell lines which express p70 beta only, resulting in appearance of high-affinity IL-2R; and blocking p55 alpha by anti-Tac mAb in cell lines which constitutively express high-affinity IL-2R eliminated both high- and low-affinity components. The existence of low, intermediate, and high IL-2R on human B cells bears important future implications for understanding the mechanism of IL-2 signaling and the role of IL-2 in B cell activation, proliferation, and differentiation.

  8. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

    PubMed Central

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates. PMID:25658443

  9. Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions.

    PubMed

    Kerr, Jason S; Wright, Gavin J

    2012-01-01

    Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods. Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t(1/2) ≤ 0.1 sec) with a low false positive rate. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters. The recombinant protein libraries are produced using a convenient and high-level mammalian expression system, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can

  10. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  11. The N-terminal domains of both NR1 and NR2 subunits determine allosteric Zn2+ inhibition and glycine affinity of N-methyl-D-aspartate receptors.

    PubMed

    Madry, Christian; Mesic, Ivana; Betz, Heinrich; Laube, Bodo

    2007-12-01

    The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1- and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn2+ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly. PMID:17878266

  12. Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states

    PubMed Central

    Little, Joshua W.; Ford, Amanda; Symons-Liguori, Ashley M.; Chen, Zhoumou; Janes, Kali; Doyle, Timothy; Xie, Jennifer; Luongo, Livio; Tosh, Dillip K.; Maione, Sabatino; Bannister, Kirsty; Dickenson, Anthony H.; Vanderah, Todd W.; Porreca, Frank; Jacobson, Kenneth A.

    2015-01-01

    Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain. PMID:25414036

  13. The Length and Flexibility of the 2-Substituent of 9-Ethyladenine Derivatives Modulate Affinity and Selectivity for the Human A2A Adenosine Receptor.

    PubMed

    Thomas, Ajiroghene; Buccioni, Michela; Dal Ben, Diego; Lambertucci, Catia; Marucci, Gabriella; Santinelli, Claudia; Spinaci, Andrea; Kachler, Sonja; Klotz, Karl-Norbert; Volpini, Rosaria

    2016-08-19

    The A2A adenosine receptor (A2A AR) is a key target for the development of pharmacological tools for the treatment of central nervous system disorders. Previous works have demonstrated that the insertion of substituents at various positions on adenine leads to A2A AR antagonists with affinity in the micromolar to nanomolar range. In this work, a series of 9-ethyladenine derivatives bearing phenylalkylamino, phenylakyloxy or phenylakylthio groups of different lengths at the 2-position were synthesised and tested against the human adenosine receptors. The derivatives showed sub-micromolar affinity for these membrane proteins. The further introduction of a bromine atom at the 8-position has the effect of improving the affinity and selectivity for all ARs and led to compounds that are able bind to the A2A AR subtype at low nanomolar levels. Functional studies confirmed that the new adenine derivatives behave as A2A AR antagonists with half-maximal inhibitory concentration values in the nanomolar range. Molecular modelling studies provide a description of the possible binding mode of these compounds at the A2A AR and an interpretation of the affinity data at this AR subtype. PMID:27037522

  14. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

    SciTech Connect

    Rannug, A.; Rannug, U.; Rosenkranz, H.S.; Winqvist, L.; Westerholm, R.; Agurell, E.; Grafstroem, A.K.

    1987-11-15

    The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with /sup 3/H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

  15. A DFT and semiempirical model-based study of opioid receptor affinity and selectivity in a group of molecules with a morphine structural core.

    PubMed

    Bruna-Larenas, Tamara; Gómez-Jeria, Juan S

    2012-01-01

    We report the results of a search for model-based relationships between mu, delta, and kappa opioid receptor binding affinity and molecular structure for a group of molecules having in common a morphine structural core. The wave functions and local reactivity indices were obtained at the ZINDO/1 and B3LYP/6-31G(∗∗) levels of theory for comparison. New developments in the expression for the drug-receptor interaction energy expression allowed several local atomic reactivity indices to be included, such as local electronic chemical potential, local hardness, and local electrophilicity. These indices, together with a new proposal for the ordering of the independent variables, were incorporated in the statistical study. We found and discussed several statistically significant relationships for mu, delta, and kappa opioid receptor binding affinity at both levels of theory. Some of the new local reactivity indices incorporated in the theory appear in several equations for the first time in the history of model-based equations. Interaction pharmacophores were generated for mu, delta, and kappa receptors. We discuss possible differences regulating binding and selectivity in opioid receptor subtypes. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process. PMID:25379287

  16. Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.

    PubMed

    Matsushima, Ayami; Nishimura, Hirokazu; Matsuyama, Yutaka; Liu, Xiaohui; Costa, Tommaso; Shimohigashi, Yasuyuki

    2016-11-01

    We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016. PMID:27271345

  17. A DFT and Semiempirical Model-Based Study of Opioid Receptor Affinity and Selectivity in a Group of Molecules with a Morphine Structural Core

    PubMed Central

    Bruna-Larenas, Tamara; Gómez-Jeria, Juan S.

    2012-01-01

    We report the results of a search for model-based relationships between mu, delta, and kappa opioid receptor binding affinity and molecular structure for a group of molecules having in common a morphine structural core. The wave functions and local reactivity indices were obtained at the ZINDO/1 and B3LYP/6-31G∗∗ levels of theory for comparison. New developments in the expression for the drug-receptor interaction energy expression allowed several local atomic reactivity indices to be included, such as local electronic chemical potential, local hardness, and local electrophilicity. These indices, together with a new proposal for the ordering of the independent variables, were incorporated in the statistical study. We found and discussed several statistically significant relationships for mu, delta, and kappa opioid receptor binding affinity at both levels of theory. Some of the new local reactivity indices incorporated in the theory appear in several equations for the first time in the history of model-based equations. Interaction pharmacophores were generated for mu, delta, and kappa receptors. We discuss possible differences regulating binding and selectivity in opioid receptor subtypes. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process. PMID:25379287

  18. Clobazam and Its Active Metabolite N-desmethylclobazam Display Significantly Greater Affinities for α2- versus α1-GABAA–Receptor Complexes

    PubMed Central

    Jensen, Henrik Sindal; Nichol, Kathryn; Lee, Deborah; Ebert, Bjarke

    2014-01-01

    Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α1-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α2 subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α1, α2, α3, or α5), β2, and γ2 subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α2- vs. α1-receptor complexes, a difference not observed for CLN, for which no distinction between α2 and α1 receptors was observed. Our experiments with ZOL confirmed the high preference for α1 receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB. PMID:24533090

  19. Analysis of D2 dopamine receptor occupancy with quantitative SPET using the high-affinity ligand [123I]epidepride: resolving conflicting findings.

    PubMed

    Erlandsson, Kjell; Bressan, Rodrigo A; Mulligan, Rachel S; Ell, Peter J; Cunningham, Vincent J; Pilowsky, Lyn S

    2003-07-01

    Recent studies of limbic cortical dopamine D(2) receptor occupancy by clozapine using high-affinity PET and SPET radioligands have produced conflicting findings. It has been suggested that these divergent findings are due to between-study differences in the method used to estimate D(2) receptor-binding potential. We compared different methods for estimating striatal and temporal cortical D(2) receptor occupancy with high-affinity tracers. In vivo experimental SPET data, obtained with [(123)I]epidepride were analysed with reference tissue kinetic modeling and with the ratio method, applied to data corresponding to short (60 min) and long (240 min) acquisition times. Dopamine D(2) receptor occupancy by the atypical antipsychotic drug risperidone was evaluated. Simulation experiments were also performed, comparing occupancy values obtained for different receptor densities in relation to different data acquisition times. The simulation results revealed that previously published data regarding errors in occupancy estimation by analysis of time activity data acquired for 60 min cannot be extrapolated to studies performed over 240 min. The ratio method provided accurate temporal cortical D(2) receptor occupancy values when applied to data from a late time period, but underestimated the occupancy with earlier data. In striatum, both the late data ratio method and reference tissue kinetic modeling using all data underestimated D(2) receptor occupancy. However, more accurate analyses of striatal D(2) occupancy still showed selective limbic/cortical occupancy by risperidone. Our results substantiate the previous [(123)I]epidepride findings of high temporal cortical occupancy by other atypical antipsychotic drugs and suggest that a potential source of conflicting findings might be short scanning times imposed by [(11)C]FLB 457, leading to underestimation of temporal cortical D(2) receptor occupancy by this method. PMID:12880845

  20. Distinction between high-affinity (/sup 3/H)phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

    SciTech Connect

    El-Fakahany, E.E.; Triggle, D.J.; Eldefrawi, A.T.; Eldefrawi, M.E.

    1984-05-01

    (/sup 3/H)Phencyclidine ((/sup 3/H)PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of (/sup 3/H)PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the un-ionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific (/sup 3/H)PCP binding was displaced effectively by several PCP analogs and Ca/sup + +/ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of (/sup 3/H)quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities.

  1. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  2. Synthesis and Structure-Affinity Relationships of Selective High-Affinity 5-HT4 Receptor Antagonists: Application to the Design of New Potential Single Photon Emission Computed Tomography (SPECT) Tracers

    PubMed Central

    Dubost, Emmanuelle; Dumas, Noé; Fossey, Christine; Magnelli, Rosa; Butt-Gueulle, Sabrina; Ballandonne, Céline; Caignard, Daniel H.; Dulin, Fabienne; de-Oliveira Santos, Jana Sopkova; Millet, Philippe; Charnay, Yves; Rault, Sylvain; Cailly, Thomas; Fabis, Frederic

    2012-01-01

    The work described herein aims at finding new potential ligands for the brain imaging of 5-HT4 receptors using single-photon emission computed tomography (SPECT). Starting from the non-substituted phenanthridine compound 4a exhibiting a Ki value of 51 nM on 5-HT4R, we explored structure-affinity in this series. We found that substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom inside the tricyclic framework led to increase both the affinity and the selectivity for 5-HT4R suggesting the design of the antagonist 4v exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT4R antagonist (1-butylpiperidin-4-yl)methyl-8-amino-7-iodo[123I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate ([123I]1, [123I]SB 207710) both in vitro and in vivo in brain. Compound 11d was radiolabeled with [125I]iodine, providing a potential SPECT candidate for brain imaging of 5-HT4R. PMID:23102207

  3. The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

    PubMed Central

    Starkl, Philipp; Bevins, Charles L.; Scheiner, Otto; Boltz-Nitulescu, George; Wrba, Fritz; Jensen-Jarolim, Erika

    2010-01-01

    Background IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium. Methodology/Principal Findings FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains. Conclusions/Significance Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested. PMID:20126404

  4. Affinity capillary electrophoresis and quantum mechanical calculations applied to the investigation of hexaarylbenzene-based receptor binding with lithium ion.

    PubMed

    Ehala, Sille; Toman, Petr; Rathore, Rajendra; Makrlík, Emanuel; Kašička, Václav

    2011-09-01

    In this study, two complementary approaches, affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations, have been employed for quantitative characterization and structure elucidation of the complex between hexaarylbenzene (HAB)-based receptor R and lithium ion Li(+) . First, by means of ACE, the apparent binding constant of LiR(+) complex (K LiR +) in methanol was determined from the dependence of the effective electrophoretic mobilities of LiR(+) complex on the concentration of lithium ions in the 25 mM Tris/50 mM chloroacetate background electrolyte (BGE) using non-linear regression analysis. Prior to regression analysis, the effective electrophoretic mobilities of the LiR(+) complex were corrected to reference temperature 25 °C and constant ionic strength 25 mM. The apparent binding constant of the LiR(+) complex in the above methanolic BGE was evaluated as logK LiR + = 1.15±0.09. Second, the most probable structures of nonhydrated LiR(+) and hydrated LiR(+)·3H(2)O complexes were derived by DFT calculations. The optimized structure of the hydrated LiR(+)·3H(2)O complex was found to be more realistic than the nonhydrated LiR(+) complex because of the considerably higher binding energy of LiR(+)·3H(2)O complex (500.4 kJ/mol) as compared with LiR(+) complex (427.5 kJ/mol). PMID:21780285

  5. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    PubMed Central

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  6. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    PubMed

    Messingham, Kelly N; Holahan, Heather M; Frydman, Alexandra S; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  7. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    PubMed

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  8. Potentials and pitfalls using high affinity radioligands in PET and SPET determinations on regional drug induced D2 receptor occupancy--a simulation study based on experimental data.

    PubMed

    Olsson, H; Farde, L

    2001-10-01

    The D2 dopamine receptor density ranges from 0.2 to 40 nM among human brain regions. For high density regions radioligands like [(11)C]raclopride provide accurate and reliable estimates of the receptor density. In research on neuropsychiatric disorders there is, however, a growing need for quantitative approaches that accurately measure D2 dopamine receptor occupancy induced by drugs or endogenous dopamine in regions with low receptor density. The new high affinity radioligands [(11)C]FLB 457 and [(123)I]epidepride have been shown to provide a signal for extrasriatal D2 dopamine receptor populations in the human brain in vivo. Initial observations indicate, however, that the time required to reach equilibrium is dependent on receptor density. Ratio analyses may thus not be readily used for comparisons among different brain regions. The aim of the present simulation study was to examine commonly used approaches for calculation of drug induced D2 dopamine receptor occupancy among regions with widely different receptor density. The input functions and the rate constants of [(11)C]FLB 457 and the reference ligand [(11)C]raclopride were first used in a simulation estimating the effect of receptor density on equilibrium time. In a second step we examined how errors produced by inaccurate determination of the binding potential parameter propagate to calculations of drug induced receptor occupancy. The simulations showed a marked effect of receptor density on equilibrium time for [(11)C]FLB 457, but not for [(11)C]raclopride. For [(11)C]FLB 457, a receptor density above about 7 nM caused the time of equilibrium to fall beyond time of data acquisition (1 h). The use of preequilibrium data caused the peak equilibrium and the end time ratio approaches but not the simplified reference tissue model (SRTM) approach to underestimate the binding potential and thus also the drug occupancy calculated for high-density regions. The study supports the use of ratio and SRTM analyses in

  9. Characterization of a Pachymedusa dacnicolor–Sauvagine Analog as a New High-Affinity Radioligand for Corticotropin-Releasing Factor Receptor Studies

    PubMed Central

    Perrin, Marilyn H.; Tan, Laura A.; Vaughan, Joan M.; Lewis, Kathy A.; Donaldson, Cynthia J.; Miller, Charleen; Erchegyi, Judit; Rivier, Jean E.

    2015-01-01

    The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [125ITyr0-Glu1, Nle17]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor–expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors. PMID:25736419

  10. Topological sub-structural molecular design (TOPS-MODE): a useful tool to explore key fragments of human A3 adenosine receptor ligands.

    PubMed

    Saíz-Urra, Liane; Teijeira, Marta; Rivero-Buceta, Virginia; Helguera, Aliuska Morales; Celeiro, Maria; Terán, Ma Carmen; Besada, Pedro; Borges, Fernanda

    2016-02-01

    Adenosine regulates tissue function by activating four G-protein-coupled adenosine receptors (ARs). Selective agonists and antagonists for A3 ARs have been investigated for the treatment of a variety of immune disorders, cancer, brain, and heart ischemic conditions. We herein present a QSAR study based on a Topological sub-structural molecular design (TOPS-MODE) approach, intended to predict the A3 ARs of a diverse dataset of 124 (94 training set/ 30 prediction set) adenosine derivatives. The final model showed good fit and predictive capability, displaying 85.1 % of the experimental variance. The TOPS-MODE approach afforded a better understanding and interpretation of the developed model based on the useful information extracted from the analysis of the contribution of different molecular fragments to the affinity. PMID:26205409

  11. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  12. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

    NASA Astrophysics Data System (ADS)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III

    2004-03-01

    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  13. Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI

    PubMed Central

    Kiyoshi, Masato; Caaveiro, Jose M.M.; Kawai, Takeaki; Tashiro, Shinya; Ide, Teruhiko; Asaoka, Yoshiharu; Hatayama, Kouta; Tsumoto, Kouhei

    2015-01-01

    Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI. PMID:25925696

  14. Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors.

    PubMed

    Donohue, Sean R; Varnäs, Katarina; Jia, Zhisheng; Gulyás, Balázs; Pike, Victor W; Halldin, Christer

    2009-11-01

    There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB1) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB1 receptor ligand ([125I]8, [125I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [125I]SD7015). By autoradiography in vitro, [125I]8 showed selective binding to CB1 receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate. PMID:19767206

  15. Syntheses of 2-amino and 2-halothiazole derivatives as high-affinity metabotropic glutamate receptor subtype 5 ligands and potential radioligands for in vivo imaging.

    PubMed

    Siméon, Fabrice G; Wendahl, Matthew T; Pike, Victor W

    2011-02-10

    The structure of the potent selective mGlu(5) ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu(5) ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed subnanomolar mGlu(5) affinity. 10b also displayed >10000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [(18)F]fluoride ion (t(1/2) = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu(5) with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu(5) and may also serve as a lead to a potential (123)I-labeled ligand for imaging brain mGlu(5) with single photon emission computed tomography. PMID:21207959

  16. Synthesis, structure and affinity of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones for CNS central and peripheral benzodiazepine receptors.

    PubMed

    Andronati, Sergey; Semenishyna, Ekaterina; Pavlovsky, Victor; Simonov, Yuriy; Makan, Svetlana; Boyko, Irina; Burenkova, Natalya; Gdaniec, Maria; Cardinael, Pascal; Bouillon, Jean-Philippe; Mazepa, Alexander

    2010-04-01

    A series of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones (7-15) was synthesized and their in vitro affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine receptor (PBR) of rat brain was studied. Racemic mixture of 7-bromo-3-(2-methoxy)ethoxy-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (13) was separated into enantiomers 14, 15 by chiral HPLC. Absolute configuration of R-enantiomer 15 was determined by the method of X-ray diffraction analysis. The affinity of S-enantiomer 14 for CBR ( IC50)=245 nM) is 20-fold higher than the affinity of R-enantiomer 15 (IC50)=4,930 nM). A high selectivity for CBR versus PBR (IC50) (PBR)>10,000 nM) was shown by all reported compounds. Compound 12 was revealed as a potent (IC50)=9 nM) and selective CBR ligand among the synthesized 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones. PMID:20061068

  17. Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.

    PubMed

    Patel, Seema; Mathonet, Pascale; Jaulent, Agnes M; Ullman, Christopher G

    2013-04-01

    WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications. PMID:23378640

  18. Syntheses of 2-Amino and 2-Halothiazole Derivatives as High-Affinity Metabotropic Glutamate Receptor Subtype 5 Ligands and Potential Radioligands for In Vivo Imaging

    PubMed Central

    Siméon, Fabrice G; Wendahl, Matthew T.; Pike, Victor W.

    2011-01-01

    The structure of the potent selective mGlu5 ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu5 ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed sub-nanomolar mGlu5 affinity. 10b also displayed >10,000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [18F]fluoride ion (t1/2 = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu5 with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu5 and may also serve as a lead to a potential 123I-labeled ligand for imaging brain mGlu5 with single photon emission computed tomography. PMID:21207959

  19. Photoaffinity labeling of mammalian. cap alpha. /sub 1/-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe. [Rats, guinea pigs, rabbits

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.; Wikberg, J.E.S.; Hagen, P.O.; DeBernardis, J.F.; Winn, M.; Arendsen, D.L.; Lefkowitz, R.J.; Caron, M.G.

    1984-02-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated ..cap alpha../sub 1/-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-(4-(5-(4-azido-3-(/sup 125/I)iodophenyl)pentanoyl)-1-piperazinyl) quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an ..cap alpha../sub 1/-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical ..cap alpha../sub 1/-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the ..cap alpha../sub 1/-adrenergic receptor.

  20. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    SciTech Connect

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-06-17

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with /sup 3/H-naloxone or /sup 3/H-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables.

  1. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcɛRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2013-05-15

    The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcɛRI) is the central protein interaction in IgE-mediated allergic reactions. The cross-linking of the IgE/FcɛRI complex, through cognate allergens, on the surface of mast cells and basophil cells results in mediator release, and thus leads to the symptoms of type I hypersensitivity responses in mammals. To develop a baseline value for subsequent equine anti-allergy drug and vaccine research, the interaction of equine IgE with its high-affinity FcɛRI receptor was investigated following the cloning and expression of equine IgE with specificity for NIP-HSA (4-hydroxy-5-iodo-3-nitrophenylacetic acid conjugated to human serum albumin). Receptor recognition and effector functions were assessed in Rat Basophil Leukemia (RBL-2H3.1) cells transfected with the α chain of equine and canine FcɛRI. Results obtained showed that the equine FcɛRI receptor recognizes both equine and canine IgE and supports similar β-hexosaminidase release levels from RBL cells transfected with equine FcɛRI, peaking at 36.68% at 100ngml(-1) antigen and 32.00% at 100ngml(-1) antigen respectively. Furthermore, the binding kinetics of the equine IgE to the equine FcɛRI receptor and the canine IgE to the same receptor was measured to be KA=6.33×10(9)M(-1) and KA=1.84×10(9)M(-1) respectively. This research established basic reagents and vitro assay systems to underpin the development of rational therapeutic intervention strategies to combat equine allergic manifestations. PMID:23485176

  2. Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

    PubMed Central

    Lin, Wan-Chen; Licht, Stuart

    2014-01-01

    High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore. PMID:25386750

  3. Different affinity windows for virus and cancer-specific T-cell receptors: implications for therapeutic strategies.

    PubMed

    Aleksic, Milos; Liddy, Nathaniel; Molloy, Peter E; Pumphrey, Nick; Vuidepot, Annelise; Chang, Kyong-Mi; Jakobsen, Bent K

    2012-12-01

    T-cell destiny during thymic selection depends on the affinity of the TCR for autologous peptide ligands presented in the context of MHC molecules. This is a delicately balanced process; robust binding leads to negative selection, yet some affinity for the antigen complex is required for positive selection. All TCRs of the resulting repertoire thus have some intrinsic affinity for an MHC type presenting an assortment of peptides. Generally, TCR affinities of peripheral T cells will be low toward self-derived peptides, as these would have been presented during thymic selection, whereas, by serendipity, binding to pathogen-derived peptides that are encountered de novo could be stronger. A crucial question in assessing immunotherapeutic strategies for cancer is whether natural TCR repertoires have the capacity for efficiently recognizing tumor-associated peptide antigens. Here, we report a comprehensive comparison of TCR affinities to a range of HLA-A2 presented antigens. TCRs that bind viral antigens fall within a strikingly higher affinity range than those that bind cancer-related antigens. This difference may be one of the key explanations for tumor immune escape and for the deficiencies of T-cell vaccines against cancer. PMID:22949370

  4. /sup 125/I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and /sup 125/I-azido-BW-A844U, a photoaffinity label

    SciTech Connect

    Patel, A.; Craig, R.H.; Daluge, S.M.; Linden, J.

    1988-06-01

    3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors; the KI for binding to A2 receptors of human platelet membranes is 2.0 microM (A2/A1 ratio = 8700). Radioiodination of the 3-aminophenethyl group resulted in 125I-BW-A844U, a radioligand that retains high affinity for A1 receptors in bovine brain membranes (KD = 0.14 nM) and to 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate-solubilized receptors (KD = 0.34 nM). Specific binding of 125I-BW-A844U represented greater than 90% of the total binding at the KD. From the association constant (K1 = 5.0 X 10(8) M-1min-1) and the dissociation constant (K-1 = 0.064 min-1), the kinetic KD (K-1/K1) in membranes was calculated to be 0.13 nM. NaCl (1 M) had little effect on the binding affinity of 125I-BW-A844U, in contrast to the large effect of salt on the binding affinity of acidic antagonist radioligands. 8-Sulfophenyltheophylline inhibited radioligand binding with a Hill coefficient of 1.0, indicative of a single affinity binding state for the antagonist. By comparison, two distinct agonist affinity states of A1 receptors for the agonist (R)-phenylisopropyladenosine could be resolved, a high affinity state (62%, KH = 74 pM) and a low affinity state (KL = 3.83 nM). The addition of 0.1 mM guanylylimidodiphosphate converted all receptors to the low affinity state. Addition of NaCl (0.5 M) decreased the fraction of receptors in the high affinity state and increased both KH and KL, suggesting that NaCl alters coupling of receptors to G proteins and influences the conformation of the receptor polypeptide, whether or not the receptor is coupled to a G protein.

  5. Biophysical Fragment Screening of the β1-Adrenergic Receptor: Identification of High Affinity Arylpiperazine Leads Using Structure-Based Drug Design

    PubMed Central

    2013-01-01

    Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein–ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively. PMID:23517028

  6. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

    PubMed Central

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T. N.; Gregory, Karen J.; Tosh, Dilip K.; Christopoulos, Arthur; Jacobson, Kenneth A.

    2016-01-01

    Biased agonism at G protein–coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias “fingerprints” for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with significant N6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5′-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  7. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor.

    PubMed

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T N; Gregory, Karen J; Tosh, Dilip K; Christopoulos, Arthur; Jacobson, Kenneth A; May, Lauren T

    2016-07-01

    Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with significant N(6) or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5'-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  8. A radioreceptor assay to study the affinity of benzodiazepines and their receptor binding activity in human plasma including their active metabolites.

    PubMed Central

    Dorow, R G; Seidler, J; Schneider, H H

    1982-01-01

    1 A radioreceptor assay has been established to measure the receptor affinities of numerous benzodiazepines in clinical use. 2 The time course of receptor binding activity was studied by this method in the plasma of eight healthy subjects randomly treated with 1 mg lormetazepam (Noctamid, 2 mg flunitrazepam (Rohypnol, and 10 mg diazepam (Valium, and placebo on a cross-over basis. Blood samples were collected up to 154 h after treatment. 3 Receptor affinities of numerous benzodiazepines on vitro show good correlation with therapeutic human doses (r = 0.96) and may be predictive of drug potency in man. 4 Mean peak plasma levels of lormetazepam binding equivalents were 4.8 +/- 1 ng/ml at 2 h after lormetazepam, 7.2 +/- 1.8 ng/ml at 8 h after flunitrazepam, and 17.9 +/- 2.7 ng/ml at 15 h after diazepam. Plasma elimination half-lives of benzodiazepine binding equivalents were 9.3, 23 and 63 h, respectively. 5 Slow elimination of benzodiazepine binding equivalents following flunitrazepam and diazepam may be due to persistent active metabolites. PMID:6121579

  9. Investigations on the 4-quinolone-3-carboxylic acid motif. 6. Synthesis and pharmacological evaluation of 7-substituted quinolone-3-carboxamide derivatives as high affinity ligands for cannabinoid receptors.

    PubMed

    Pasquini, Serena; De Rosa, Maria; Ligresti, Alessia; Mugnaini, Claudia; Brizzi, Antonella; Caradonna, Nicola P; Cascio, Maria Grazia; Bolognini, Daniele; Pertwee, Roger G; Di Marzo, Vincenzo; Corelli, Federico

    2012-12-01

    Within our studies on structure-activity relationships of 4-quinolone-3-carboxamides as cannabinoid ligands, a new series of compounds characterized by a fluoro or phenylthio group at 7-position and different substituents at N1 and carboxamide nitrogen were synthesized and evaluated for their binding ability to cannabinoid type 1 (CB1) and type 2 (CB2) receptors. Most of the compounds showed affinity for one or both cannabinoid receptors at nanomolar concentration, with K(i)(CB1) and K(i)(CB2) values ranging from 2.45 to >10,000 nM and from 0.09 to 957 nM, respectively. The N-(3,4-dichlorobenzyl)amide derivatives 27 and 40 displayed relatively low affinity, but high selectivity towards the CB1 receptor. Compounds 4 and 40, a CB2 and a CB1 ligand, respectively, behaved as partial agonists in the [(35)S]GTPγS assay. They showed very low permeability through (MDCK-MDR1) cells and might, therefore, represent possible lead structures for further optimization in the search for cannabinoid ligands unable to cross the blood-brain barrier. PMID:23085772

  10. Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.

    PubMed

    Di Marco, E; Mathor, M; Bondanza, S; Cutuli, N; Marchisio, P C; Cancedda, R; De Luca, M

    1993-10-25

    Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells. PMID:7693679

  11. Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion*

    PubMed Central

    Ziegler, Amber N.; Chidambaram, Shravanthi; Forbes, Briony E.; Wood, Teresa L.; Levison, Steven W.

    2014-01-01

    The objective of this study was to employ genetically engineered IGF-II analogs to establish which receptor(s) mediate the stemness promoting actions of IGF-II on mouse subventricular zone neural precursors. Neural precursors from the subventricular zone were propagated in vitro in culture medium supplemented with IGF-II analogs. Cell growth and identity were analyzed using sphere generation and further analyzed by flow cytometry. F19A, an analog of IGF-II that does not bind the IGF-2R, stimulated an increase in the proportion of neural stem cells (NSCs) while decreasing the proportion of the later stage progenitors at a lower concentration than IGF-II. V43M, which binds to the IGF-2R with high affinity but which has low binding affinity to the IGF-1R and to the A isoform of the insulin receptor (IR-A) failed to promote NSC growth. The positive effects of F19A on NSC growth were unaltered by the addition of a functional blocking antibody to the IGF-1R. Altogether, these data lead to the conclusion that IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R. PMID:24398690

  12. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D/sub 2/ receptor

    SciTech Connect

    Borgundvaag, B.; George, S.R.

    1985-07-29

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of (/sup 3/H)-ATP to (/sup 3/H)-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC/sub 50/ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC/sub 50/ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D/sub 2/ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table.

  13. Development of new peptide-based receptor of fluorescent probe with femtomolar affinity for Cu(+) and detection of Cu(+) in Golgi apparatus.

    PubMed

    Jung, Kwan Ho; Oh, Eun-Taex; Park, Heon Joo; Lee, Keun-Hyeung

    2016-11-15

    Developing fluorescent probes for monitoring intracellular Cu(+) is important for human health and disease, whereas a few types of their receptors showing a limited range of binding affinities for Cu(+) have been reported. In the present study, we first report a novel peptide receptor of a fluorescent probe for the detection of Cu(+). Dansyl-labeled tripeptide probe (Dns-LLC) formed a 1:1 complex with Cu(+) and showed a turn-on fluorescent response to Cu(+) in aqueous buffered solutions. The dissociation constant of Dns-LLC for Cu(+) was determined to be 12 fM, showing that Dns-LLC had more potent binding affinity for Cu(+) than those of previously reported chemical probes for Cu(+). The binding mode study showed that the thiol group of the peptide receptor plays a critical role in potent binding with Cu(+) and the sulfonamide and amide groups of the probe might cooperate to form a complex with Cu(+). Dns-LLC detected Cu(+) selectively by a turn-on response among various biologically relevant metal ions, including Cu(2+) and Zn(2+). The selectivity of the peptide-based probe for Cu(+) was strongly dependent on the position of the cysteine residue in the peptide receptor part. The fluorescent peptide-based probe penetrated the living RKO cells and successfully detected Cu(+) in the Golgi apparatus in live cells by a turn-on response. Given the growing interest in imaging Cu(+) in live cells, a novel peptide receptor of Cu(+) will offer the potential for developing a variety of fluorescent probes for Cu(+) in the field of copper biochemistry. PMID:27208475

  14. High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence.

    PubMed

    Wang, Yimeng; Sundling, Christopher; Wilson, Richard; O'Dell, Sijy; Chen, Yajing; Dai, Kaifan; Phad, Ganesh E; Zhu, Jiang; Xiao, Yongli; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T; Li, Yuxing

    2016-05-01

    Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates. PMID:27001953

  15. A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 degrees C and in ileum at 29 degrees C and 37 degrees C.

    PubMed Central

    Barlow, R B; Berry, K J; Glenton, P A; Nilolaou, N M; Soh, K S

    1976-01-01

    1 The affinity of 17 compounds for muscarine-sensitive acetylcholine receptors in atrial pacemaker cells and ileum of the guinea-pig has been measured at 29 degrees C in Ringer-Locke solution. Measurements were also made at 37 degrees C with 7 of them. 2 Some of the compounds had much higher affinity for the receptors in the ileum than for those in the atria. For the most selective compound, 4-diphenylacetoxy-N-methylpiperidine methiodide, the difference was approximately 20-fold. The receptors in the atria are therefore different the structure from those in the ileum. 3 The effect of temperature on affinity are not the same for all the compounds, tested indicating different enthalpies and entropies of adsorption and accounting for some of the difficulty experienced in predicting the affinity of new compounds. PMID:1000135

  16. Mutant LV(476-7)AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity.

    PubMed

    Alam, Jahangir; Yamato, Ichiro; Arai, Satoshi; Saijo, Shinya; Mizutani, Kenji; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki; Iwata, So; Kakinuma, Yoshimi; Murata, Takeshi

    2013-01-01

    Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV(476-7)AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity. PMID:24404436

  17. Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

    PubMed Central

    Kahn, N N; Bauman, W A; Sinha, A K

    1996-01-01

    Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI. PMID:8552614

  18. A1-, A2A- and A3-subtype adenosine receptors modulate intraocular pressure in the mouse

    PubMed Central

    Avila, Marcel Y; Stone, Richard A; Civan, Mortimer M

    2001-01-01

    Despite the potential importance of the mouse in studying the pharmacology of aqueous dynamics, measurement of intraocular pressure (IOP) in its very small eye has been problematic. Utilizing a novel servo-null electrophysiologic approach recently applied to the mouse, we have identified a diversity of adenosine-receptor mechanisms in modulating IOP in this species. We report the first evidence that A3 receptors increase IOP in any species, and verify in the mouse reports with larger mammals that A1 receptors lower and A2A receptors increase IOP. PMID:11564641

  19. Analogs of JHU75528, a PET ligand for imaging of cerebral cannabinoid receptors (CB1): development of ligands with optimized lipophilicity and binding affinity

    PubMed Central

    Fan, Hong; Kotsikorou, Evangelia; Hoffman, Alexander F.; Ravert, Hayden T.; Holt, Daniel; Hurst, Dow P.; Lupica, Carl R.; Reggio, Patricia H.; Dannals, Robert F.; Horti, Andrew G.

    2009-01-01

    Cyano analogs of Rimonabant with high binding affinity for the cerebral cannabinoid receptor (CB1) and with optimized lipophilicity have been synthesized as potential positron emission tomography (PET) ligands. The best ligands of the series are optimal targets for the future radiolabeling with PET isotopes and in vivo evaluation as radioligands with enhanced properties for PET imaging of CB1 receptors in human subjects. Extracellular electrophysiological recordings in rodent brain slices demonstrated that JHU75528, 4, the lead compound of the new series, has functional CB antagonist properties that are consistent with its structural relationship to Rimonabant. Molecular modeling analysis revealed an important role of the binding of the cyano-group with the CB1 binding pocket. PMID:18511157

  20. (I-125) 17. cap alpha. -Iodovinyl 11. beta. -methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical

    SciTech Connect

    Jagoda, E.M.; Gibson, R.E.; Goodgold, H.; Ferreira, N.; Francis, B.E.; Reba, R.C.; Rzeszotarski, W.J.; Eckelman, W.C.

    1984-04-01

    17 ..cap alpha..-(/sup 125/I)Iodovinyl 11 ..beta..-methoxyestradiol ((I-125)MIVE/sub 2/) has been prepared with high specific activity (155-2000 Ci/mmol) and a high affinity for the estrogen receptor. In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The radioactive labeling of MIVE/sub 2/ is sufficiently rapid so that (I-123)MIVE/sub 2/ has been synthesized and is currently in clinical trials. These results suggest that MIVE/sub 2/ would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

  1. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  2. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

    EPA Science Inventory

    The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

  3. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  4. High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

    PubMed

    Prasad, Joni M; Young, Patricia A; Strickland, Dudley K

    2016-08-26

    The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor. PMID:27402839

  5. Local intersection volume: a new 3D descriptor applied to develop a 3D-QSAR pharmacophore model for benzodiazepine receptor ligands.

    PubMed

    Verli, Hugo; Albuquerque, Magaly Girão; Bicca de Alencastro, Ricardo; Barreiro, Eliezer J

    2002-03-01

    In this work, we have developed a new descriptor, named local intersection volume (LIV), in order to compose a 3D-QSAR pharmacophore model for benzodiazepine receptor ligands. The LIV can be classified as a 3D local shape descriptor in contraposition to the global shape descriptors. We have selected from the literature 49 non-benzodiazepine compounds as a training data set and the model was obtained and evaluated by genetic algorithms (GA) and partial least-squares (PLS) methods using LIVs as descriptors. The LIV 3D-QSAR model has a good predictive capacity according the cross-validation test by "leave-one-out" procedure (Q(2)=0.72). The developed model was compared to a comprehensive and extensive SAR pharmacophore model, recently proposed by Cook and co-workers, for benzodiazepine receptor ligands [J. Med. Chem. 43 (2000) 71]. It showed a relevant correlation with the pharmacophore groups pointed out in that work. Our LIV 3D-QSAR model was also able to predict affinity values for a series of nine compounds (test data set) that was not included into the training data set. PMID:11900866

  6. Mutations at Beta N265 in γ-Aminobutyric Acid Type A Receptors Alter Both Binding Affinity and Efficacy of Potent Anesthetics

    PubMed Central

    Stewart, Deirdre S.; Pierce, David W.; Hotta, Mayo; Stern, Alex T.; Forman, Stuart A.

    2014-01-01

    Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α− inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15′) on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the βN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between βN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1β2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the β-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1β2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive βN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1β2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in β+/α− interfaces, we found that βN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. βN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics β+/α− anesthetic site occupancy. Our results show that βN265M mutations dramatically reduce the efficacy/transduction of

  7. Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Vacek, Antonín; Streitová, Denisa

    2007-07-01

    The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions. PMID:17383145

  8. The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

    SciTech Connect

    Cauvin, A.; Buscail, L.; Gourlet, P.; De Neef, P.; Gossen, D.; Arimura, A.; Miyata, A.; Coy, D.H.; Robberecht, P.; Christophe, J. )

    1990-07-01

    We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version) to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. ({sup 125}I)PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited ({sup 125}I)PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of ({sup 125}I)PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.

  9. Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions.

    PubMed

    Bonzi, Jeremy; Bornet, Olivier; Betzi, Stephane; Kasper, Brian T; Mahal, Lara K; Mancini, Stephane J; Schiff, Claudine; Sebban-Kreuzer, Corinne; Guerlesquin, Francoise; Elantak, Latifa

    2015-01-01

    Galectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions. PMID:25708191

  10. Alternative mRNA splicing of SMRT creates functional diversity by generating corepressor isoforms with different affinities for different nuclear receptors.

    PubMed

    Goodson, Michael L; Jonas, Brian A; Privalsky, Martin L

    2005-03-01

    Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts. PMID:15632172

  11. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

    PubMed

    Ciesla, L; Okine, M; Rosenberg, A; Dossou, K S S; Toll, L; Wainer, I W; Moaddel, R

    2016-01-29

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  12. Evaluation of adhesion force and binding affinity of phytohemagglutinin erythroagglutinating to EGF receptor on human lung cancer cells.

    PubMed

    Kuo, W-T; Dong, G-C; Yao, C-H; Huang, J-Y; Lin, F-H

    2013-01-01

    PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8), respectively, that could evaluate binding affinity. The result showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that could be immobilized on the drug carrier to guide therapeutic particles to the tumor site. PMID:23394551

  13. Characterization and affinity cross-linking of receptors for human recombinant lymphotoxin (tumor necrosis factor-beta) on a human histiocytic lymphoma cell line, U-937

    SciTech Connect

    Stauber, G.B.; Aggarwal, B.B.

    1989-02-25

    Recombinant human lymphotoxin (rhLT) expressed in a mammalian cell line was purified and used to examine its receptors on the human histiocytic lymphoma cell line U-937. rhLT was radioiodinated by the IODO-GEN method to a specific activity of 60 microCi/micrograms; the labeled protein was biologically active in the cytolytic assay, and displaceable binding to U-937 cells was observed. The specific binding reached a plateau within 10, 60, and 180 min at 37, 23, and 4 degrees C, respectively. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 0.6 nM and a capacity of 33,000 +/- 7,000 binding sites/cell. The binding of 125I-rhLT to U-937 cells could be inhibited by excess unlabeled rhLT or recombinant human tumor necrosis factor (rhTNF), suggesting a common receptor for both molecules. As competitive inhibitor of the binding, rhTNF was equal in its potency to rhLT. Bacterial derived rhLT lacking carbohydrate was also found equipotent to cell line-derived rhLT for cell binding, indicating that carbohydrate plays no significant role in receptor interaction. Additionally, 125I-rhLT was covalently attached to the cell surface via a bifunctional cross-linking reagent, ethylene glycol bis(succinimidyl succinate), solubilized, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cross-linking of the receptor to rhLT revealed two distinct bands at approximate molecular masses of 100,000 and 120,000 daltons. Both bands were absent when unlabeled rhLT or rhTNF was used for competition, indicating the specificity. Affinity cross-linking of U-937 cells with 125I-rhTNF, however, provided only a single band with a molecular mass of about 100,000 daltons. These results suggest that the manner in which rhLT interacts with its receptor is perhaps somewhat different from that of rhTNF.

  14. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    SciTech Connect

    Tiberi, M.; Magnan, J. )

    1990-05-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).

  15. Interactions of dopamine agonists with brain D1 receptors labeled by /sup 3/H-antagonists. Evidence for the presence of high and low affinity agonist-binding states

    SciTech Connect

    Leff, S.E.; Hamblin, M.W.; Creese, I.

    1985-02-01

    The interactions of dopaminergic agonists and antagonists with /sup 3/H-antagonist labeled D1 dopamine receptors of rat striatum have been characterized. (/sup 3/H)Flupentixol has been found to selectively label D1 dopamine receptors when its binding to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone. Antagonist//sup 3/H-antagonist competition curves are of uniformly steep slope (nH . 1.0) suggesting the presence of a single D1 dopamine receptor. Agonist//sup 3/H-antagonist competition curves are extremely shallow (nH less than or equal to 0.5) for agonists of high relative efficacy, suggesting the presence of heterogeneous populations of agonist-binding states of the D1 dopamine receptor. Computer-modeling techniques were used to estimate affinities and relative site densities for these heterogeneous binding states. This analysis indicates that the ratio of agonist affinities for low and high affinity agonist-binding states is correlated with agonist relative efficacies in activating adenylate cyclase in membrane homogenates. Under the assay conditions employed, the addition of saturating concentrations of guanine nucleotides reduced, but did not abolish, the relative density of high affinity agonist-binding sites. These binding data can, at least in part, be explained by postulating two states of the D1 dopamine receptor, inducible by agonists but not by antagonists and modulated by guanine nucleotides.

  16. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

    PubMed

    Streitová, D; Sefc, L; Savvulidi, F; Pospísil, M; Holá, J; Hofer, M

    2010-01-01

    Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration. PMID:19249907

  17. The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain.

    PubMed

    de Paulis, Tomas

    2003-01-01

    [(123)I]Epidepride, [(18)F]fallypride, and [(76)Br]isoremoxipride (FLB-457) and their corresponding [(11)C]labeled derivatives belong to a class of high-affinity radioligands for SPECT or PET imaging of dopamine D(2) receptors in the human brain. In contrast to previously used imaging agents, these ligands are capable of identifying extrastriatal dopamine D(2) receptors. The design of these substituted benzamides derive its origin from the atypical antipsychotic agent, remoxipride. Starting in the late 1970's, halogenated analogs of (S)-sulpiride were evaluated in binding assays and behavioral studies, leading to the discovery of remoxipride. Remoxipride was 10 times weaker than sulpiride in vitro but 50 times more potent in vivo. Search for a putative active metabolite of remoxipride led to the discovery of raclopride and eticlopride, the former becoming a useful radioligand as tritium or carbon-11 labeled form for receptor binding and PET studies, respectively. In the US, the mono-iodine analog of raclopride, [(123)I]iodobenzamide (IBZM), was found to have moderate putamen-to-cerebellum ratio in rat and human brain. Continued search for metabolites of remoxipride led to the discovery of its 3,6-dihydroxy derivative, NCQ-344, with an extremely potent in vivo activity in the rat. SAR studies of the metabolites of remoxipride led to the discovery of the 3-methoxy isomer, isoremoxipride (FLB-457) and its corresponding 6-hydroxy analog, FLB-463, both having affinities for the dopamine D(2) receptor in the 20-30 pM range. Later, the 5-[(123)I]iodo analog of FLB-463, [(123)I]ioxipride ([(123)I]NCQ-298), became a potential SPECT imaging agent. In the mean time, the deshydroxy analog of IBZM, [(125)I]iodopride, showed binding potential in the rat similar to [(125)I]IBZM. Epidepride was designed by combining the structure of isoremoxipride with that of iodopride. In 1988, epidepride was independently prepared and radiolabeled in three separate laboratories in Stockholm

  18. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  19. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

    PubMed Central

    Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  20. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  1. High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

    PubMed

    Lankisch, T O; Nozu, F; Owyang, C; Tsunoda, Y

    1999-09-01

    In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic

  2. IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

    SciTech Connect

    Rodan, S.B.; Wesolowski, G.; Chin, J.; Limjuco, G.A.; Schmidt, J.A.; Rodan, G.A. )

    1990-08-15

    IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.

  3. Role of the low-affinity glucocorticoid receptor in the regulation of behavior and energy metabolism in the migratory red knot Calidris canutus islandica.

    PubMed

    Landys, Meta M; Piersma, Theunis; Ramenofsky, Marilyn; Wingfield, John C

    2004-01-01

    Plasma corticosterone increases in association with migratory flight in the red knot Calidris canutus islandica, suggesting that corticosterone may promote migratory activity and/or energy mobilization in this species. This hypothesis is supported by general effects of glucocorticoids, which include stimulation of locomotion and the mobilization of energy depots. We experimentally examined the role of elevated corticosterone levels in the migratory red knot by comparing foraging behavior, flight frequency, and plasma metabolites between vehicle-injected controls and birds treated with RU486, an antagonist to the genomic low-affinity glucocorticoid receptor (GR). We predicted that RU486 treatment would interfere with energy mobilization. However, we expected no effects on flight activity because recent studies suggest that glucocorticoids affect locomotion through a nongenomic receptor. Finally, because glucocorticoids exert permissive effects on food intake, we postulated that RU486 treatment in the red knot would interfere with feeding. Results were consistent with the latter prediction, suggesting that the GR participates in the promotion of hyperphagia, the intense feeding state that is characteristic of the migratory condition. RU486 treatment did not affect flight frequency, suggesting that corticosterone may support migratory activity through a receptor other than the GR. Energy metabolism (as determined through plasma metabolites) was also unaffected by RU486, possibly because energetic demands experienced by captive birds were low. PMID:15449237

  4. Tritiation of delta opioid-receptor selective antagonist dipeptide ligands with extraordinary affinity containing 2', 6'dimethyltyrosine

    NASA Astrophysics Data System (ADS)

    Kertész, I.; Tóth, G.; Balboni, G.; Guerrini, R.; Salvadori, S.

    1999-01-01

    Recently a new class of δ opioid antagonists has been discovered by using Tyr-Tic sequence. The substitution of Tyr1 by Dmt resulted in a new analogue (H-Dmt-Tic-OH) with enhanced affinity and selectivity. Because of its excellent property we chose it for labelling with tritium. At the same time peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, and they lose some of their binding ability. To avoid this unwanted side-reaction we synthetized the N-methylated analogue (N,N(Me)2-Dmt-Tic-OH), and it was more stable under storage condition, but δ affinity declined moderately. On the basis of this information we prepared diiodinated analogues of these dipeptides. Catalytic dehalotritiation of precursors resulted in tritiated peptides. High specific radioactivity, 44.67 Ci/mmol with [3H]Dmt-Tic-OH and 59.88 Ci/mmol with N,N(Me)2-[3H]Dmt-Tic-OH were achieved.

  5. Tritiation of delta opioid-receptor selective antagonist dipeptide ligands with extraordinary affinity containing 2‧, 6‧dimethyltyrosine

    NASA Astrophysics Data System (ADS)

    Kertész, I.; Tóth, G.; Balboni, G.; Guerrini, R.; Salvadori, S.

    1999-01-01

    Recently a new class of δ opioid antagonists has been discovered by using Tyr-Tic sequence. The substitution of Tyr1 by Dmt resulted in a new analogue (H-Dmt-Tic-OH) with enhanced affinity and selectivity. Because of its excellent property we chose it for labelling with tritium. At the same time peptides containing Tic at position 2 undergo spontaneous diketopiperazine formation in some solvents, and they lose some of their binding ability. To avoid this unwanted side-reaction we synthetized the N-methylated analogue (N,N(Me)2-Dmt-Tic-OH), and it was more stable under storage condition, but δ affinity declined moderately. On the basis of this information we prepared diiodinated analogues of these dipeptides. Catalytic dehalotritiation of precursors resulted in tritiated peptides. High specific radioactivity, 44.67 Ci/mmol with [3H]Dmt-Tic-OH and 59.88 Ci/mmol with N,N(Me)2-[3H]Dmt-Tic-OH were achieved.

  6. Functional role for the angiotensin II receptor (AT1A) 3'-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins.

    PubMed Central

    Thekkumkara, T J; Thomas, W G; Motel, T J; Baker, K M

    1998-01-01

    cells with and without the 3'-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3'-UTR, identifying a role for the 3'-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3'-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3'-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3'-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3'-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3'-UTR. Our data suggest that the 3'-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3'-UTR by RNA-binding proteins. PMID:9425107

  7. The mouse lp(A3)/Edg7 lysophosphatidic acid receptor gene: genomic structure, chromosomal localization, and expression pattern.

    PubMed

    Contos, J J; Chun, J

    2001-04-18

    The extracellular signaling molecule, lysophosphatidic acid (LPA), mediates proliferative and morphological effects on cells and has been proposed to be involved in several biological processes including neuronal development, wound healing, and cancer progression. Three mammalian G protein-coupled receptors, encoded by genes designated lp (lysophospholipid) receptor or edg (endothelial differentiation gene), mediate the effects of LPA, activating similar (e.g. Ca(2+) release) as well as distinct (neurite retraction) responses. To understand the evolution and function of LPA receptor genes, we characterized lp(A3)/Edg7 in mouse and human and compared the expression pattern with the other two known LPA receptor genes (lp(A1)/Edg2 and lp(A2)/Edg4non-mutant). We found mouse and human lp(A3) to have nearly identical three-exon genomic structures, with introns upstream of the coding region for transmembrane domain (TMD) I and within the coding region for TMD VI. This structure is similar to lp(A1) and lp(A2), indicating a common ancestral gene with two introns. We localized mouse lp(A3) to distal Chromosome 3 near the varitint waddler (Va) gene, in a region syntenic with the human lp(A3) chromosomal location (1p22.3-31.1). We found highest expression levels of each of the three LPA receptor genes in adult mouse testes, relatively high expression levels of lp(A2) and lp(A3) in kidney, and moderate expression of lp(A2) and lp(A3) in lung. All lp(A) transcripts were expressed during brain development, with lp(A1) and lp(A2) transcripts expressed during the embryonic neurogenic period, and lp(A3) transcript during the early postnatal period. Our results indicate both overlapping as well as distinct functions of lp(A1), lp(A2), and lp(A3). PMID:11313151

  8. Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

    PubMed Central

    Ware, J; Dent, J A; Azuma, H; Sugimoto, M; Kyrle, P A; Yoshioka, A; Ruggeri, Z M

    1991-01-01

    von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes. Images PMID:2011604

  9. Early detection of influenza A(H5) viruses with affinity for the human sialic acid receptor by MALDI-TOF mass spectrometry based mutation detection.

    PubMed

    Yea, C; McCorrister, S; Westmacott, G; Petric, M; Tellier, R

    2011-03-01

    Highly pathogenic avian influenza (HPAI) A(H5N1) strains have been causing sporadic cases of disease in South East Asia and Africa for many years. These cases are associated with a high fatality rate, and it is feared that the virus could evolve into a strain capable of causing a pandemic. It is likely that a requirement for a A(H5) pandemic to occur is a switch in the receptor affinity of the virus. Candidate mutations in the hemagglutinin glycoprotein have been identified in the literature, and their emergence in circulating viruses would be an ominous development. This study describes a method to identify the presence of these mutations, even within a quasispecies, using RT-PCR followed by in vitro translation and peptide characterization by MALDI-TOF mass spectrometry. PMID:21195111

  10. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    PubMed

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves. PMID:27129432

  11. Amino-substituted 1,8-naphthyridines and pyrido[2,3-d]pyrimidines: new compounds with affinity for A1- and A2-adenosine receptors.

    PubMed

    Müller, C E; Grahner, B; Heber, D

    1994-12-01

    Two novel classes of adenosine receptor (AR) antagonists, 4-amino-1,8-naphthyridines and 5-aminopyrido[2,3-d]pyrimidines, have been identified and investigated in radioligand binding assays. The compounds exhibit affinities for A1 and A2a AR of rat brain in the micromolar range. 1,8-Naphthyridines are non-selective, or somewhat selective for either A1- or A2 AR. Pyrido[2,3-d]pyrimidines are several-fold selective for A1 AR, the most potent and selective compound being 5-n-butylamino-1,3-dimethyl-1,2,3,4-tetrahydropyrido-[2,3-d]pyr imi dine-2,4-dione (12) with a Ki value of 1.8 microM at A1 AR and greater than 10-fold A1-selectivity. PMID:7838877

  12. EP 171: a high affinity thromboxane A2-mimetic, the actions of which are slowly reversed by receptor blockade.

    PubMed Central

    Jones, R. L.; Wilson, N. H.; Lawrence, R. A.

    1989-01-01

    1. Replacement of the four-carbon omega-terminus in 9,11-endoxy-10a-homo prostaglandin H2 with a p-fluorophenoxy group produces a compound (EP 171) with very high agonist potency at TP-receptors. 2. On six isolated smooth muscle preparations EP 171 was 33-167 times more potent as a TP-receptor agonist than U-46619 (11,9-epoxymethano PGH2); EC50 values ranged from 45 to 138 pM. The actions of EP 171 were difficult to study because of their slow onset and offset. For example, on the guinea-pig trachea the time required for 50% reversal of EP 171-induced contractions during washout was about 3 h. 3. On the pig pulmonary artery, a more rapidly responding preparation, it was possible to show that the TP-receptor antagonist EP 092 blocked the contractile actions of EP 171 and U-46619 to similar extents: pA2 = 8.09 and 8.15 respectively. 4. EP 171 was also a very potent activator of human blood platelets, being about 90 times more potent than U-46619. Both shape change (0.1 nM) and aggregation (1 nM) were slow in onset, a profile not previously observed for a thromboxane A2-mimetic. 5. When potencies at TP-, EP1-(guinea-pig fundus) and FP-(dog iris sphincter) receptors were compared, EP 171 showed a higher specificity as a TP-receptor agonist than either STA2 or U-46619. These studies also showed that contrary to earlier reports, the guinea-pig fundus does contain TP-receptors mediating muscle contraction. However, the maximal response due to activation of TP-receptors was only about 35% of the PGE2 maximum. 6. Established responses to EP 171 were slowly reversed following addition of a high concentration of a TP-receptor antagonist (EP 092, GR 32191 or BM 13177). Faster reversals of three less potent 16-p-halophenoxy prostanoids and U-46619 were obtained. Half-times for offset (and onset) of agonist action appeared to correlate with potency rather than with lipophilicity. 7. Competition between the agonists and a radio iodinated PTA2 derivative ([125I]-PTA-OH) for

  13. Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor

    SciTech Connect

    Ware, J.; Dent, J.A.; Azuma, Hiroyuki; Sugimoto, Mitsuhiko; Kyrle, P.A.; Yoshioka, Akira; Ruggeri, Z.M. )

    1991-04-01

    von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. The authors have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp{sup 550} to Cys{sup 550}, located in the GP IB-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp{sup 550} or a mutant Cys{sup 550} residue, bound directly to GP Ib in the absence of modulators and with similar affinity. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

  14. Defect in the membrane expression of high affinity 72-kD Fc gamma receptors on phagocytic cells in four healthy subjects.

    PubMed Central

    Ceuppens, J L; Baroja, M L; Van Vaeck, F; Anderson, C L

    1988-01-01

    Three different receptors for the Fc portion of IgG (FcR) have been characterized on human leukocytes. We have identified four healthy members of one family, whose blood phagocytic cells lack functional 72 kD high-affinity FcRI. Their monocytes were unable to bind the Fc portion of mouse (m)-IgG2a and of monomeric human IgG, and they were unreactive with two anti-FcRI monoclonal antibodies. Thus, FcRI is either absent, expressed at very low density, or is so structurally altered as to be unable to bind both its ligand and the anti-FcRI antibodies. The failure to bind the Fc portion of mIgG2a underlies the previously reported inability of these monocytes to support T cell mitogenesis on OKT3 stimulation. FcRI was not inducible upon incubation of their monocytes or neutrophils in gamma interferon. However, their monocytes were able to bind aggregated human IgG, and to phagocytose IgG-coated particles in vitro. Both functions could be blocked with a monoclonal antibody to the 40-kD low-affinity FcRII and therefore apparently were mediated exclusively through FcRII. This also demonstrates that FcRII can mediate phagocytosis independently. Despite the FcRI defect, these subjects had no circulating immune complexes, no evidence of autoimmune pathology and no increased susceptibility to infections. PMID:2969920

  15. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle.

    PubMed

    Ho, S M; Fehrer, S; Yu, M; Liang, L C; Press, D

    1988-06-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species. PMID:3417113

  16. Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

    PubMed Central

    Gupta, Shashi; Hirota, Masao; Waugh, Sheela M.; Murakami, Ikuo; Suzuki, Tomoki; Muraguchi, Masahiro; Shibamori, Masafumi; Ishikawa, Yuichi; Jarvis, Thale C.; Carter, Jeffrey D.; Zhang, Chi; Gawande, Bharat; Vrkljan, Michael; Janjic, Nebojsa; Schneider, Daniel J.

    2014-01-01

    Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2′-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases. PMID:24415766

  17. High-affinity binding of (/sup 3/H)estradiol-17 beta by an estrogen receptor in the liver of the turtle

    SciTech Connect

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-06-01

    Specific (3H)estradiol-17 beta ((3H)E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds (3H)E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of (3H)E2 binding activity in both cytosolic and nuclear fractions. The exchange between (3H)E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species.

  18. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml(-1) of antigen. This assay was modified from previous assays used to study human and canine allergic responses. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease. PMID:25406512

  19. Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity and functionality.

    PubMed

    Osman, Noha A; Ligresti, Alessia; Klein, Christian D; Allarà, Marco; Rabbito, Alessandro; Di Marzo, Vincenzo; Abouzid, Khaled A; Abadi, Ashraf H

    2016-10-21

    CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki = 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (∼70 and ∼200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators. PMID:27448919

  20. Blockage of A2A and A3 adenosine receptors decreases the desensitization of human GABAA receptors microtransplanted to Xenopus oocytes

    PubMed Central

    Roseti, Cristina; Palma, Eleonora; Martinello, Katiuscia; Fucile, Sergio; Morace, Roberta; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonietta; Giangaspero, Felice; Aronica, Eleonora; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Cristalli, Gloria; Lambertucci, Catia; Marucci, Gabriella; Volpini, Rosaria; Limatola, Cristina; Eusebi, Fabrizio

    2009-01-01

    We previously found that the endogenous anticonvulsant adenosine, acting through A2A and A3 adenosine receptors (ARs), alters the stability of currents (IGABA) generated by GABAA receptors expressed in the epileptic human mesial temporal lobe (MTLE). Here we examined whether ARs alter the stability (desensitization) of IGABA expressed in focal cortical dysplasia (FCD) and in periglioma epileptic tissues. The experiments were performed with tissues from 23 patients, using voltage-clamp recordings in Xenopus oocytes microinjected with membranes isolated from human MTLE and FCD tissues or using patch-clamp recordings of pyramidal neurons in epileptic tissue slices. On repetitive activation, the epileptic GABAA receptors revealed instability, manifested by a large IGABA rundown, which in most of the oocytes (≈70%) was obviously impaired by the new A2A antagonists ANR82, ANR94, and ANR152. In most MTLE tissue-microtransplanted oocytes, a new A3 receptor antagonist (ANR235) significantly improved IGABA stability. Moreover, patch-clamped pyramidal neurons from human neocortical slices of periglioma epileptic tissues exhibited altered IGABA rundown on ANR94 treatment. Our findings indicate that antagonizing A2A and A3 receptors increases the IGABA stability in different epileptic tissues and suggest that adenosine derivatives may offer therapeutic opportunities in various forms of human epilepsy. PMID:19721003

  1. Polyamidoamine (PAMAM) Dendrimer Conjugates of Clickable Agonists of the A3 Adenosine Receptor and Coactivation of the P2Y14 Receptor by a Tethered Nucleotide

    SciTech Connect

    Tosh, Dilip, K.; Yoo, Lena S.; Chinn, Moshe; Hong, Kunlun; Kilbey, II, S Michael; Barrett, Matthew O.; Fricks, Ingrid P.; Harden, T. Kendall; Jacobson, Kenneth A.

    2010-01-01

    We previously synthesized a series of potent and selective A{sub 3} adenosine receptor (AR) agonists (North-methanocarba nucleoside 5{prime}-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed 'click' chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A{sub 3}AR activation was preserved in these multivalent conjugates, which bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A{sub 3}AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A{sub 3} and P2Y{sub 14} receptors (via amide-linked uridine-5{prime}-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

  2. Gene-environment interactions affect long-term depression (LTD) through changes in dopamine receptor affinity in Snap25 deficient mice

    PubMed Central

    Baca, Michael; Allan, Andrea M.; Partridge, L. Donald; Wilson, Michael C.

    2013-01-01

    Genes and environmental conditions interact in the development of cognitive capacities and each plays an important role in neuropsychiatric disorders such as attention deficit/hyperactivity disorder (ADHD) and schizophrenia. Multiple studies have indicated that the gene for the SNARE protein SNAP-25 is a candidate susceptibility gene for ADHD, as well as schizophrenia, while maternal smoking is a candidate environmental risk factor for ADHD. We utilized mice heterozygous for a Snap25 null allele and deficient in SNAP-25 expression to model genetic effects in combination with prenatal exposure to nicotine to explore genetic and environmental interactions in synaptic plasticity and behavior. We show that SNAP-25 deficient mice exposed to prenatal nicotine exhibit hyperactivity and deficits in social interaction. Using a high frequency stimulus electrophysiological paradigm for long-term depression (LTD) induction, we examined the roles of dopaminergic D2 receptors (D2Rs) and cannabinoid CB1 receptors (CB1Rs), both critical for LTD induction in the striatum. We found that prenatal exposure to nicotine in Snap25 heterozygote null mice produced a deficit in the D2R-dependent induction of LTD, although CB1R regulation of plasticity was not impaired. We also show that prenatal nicotine exposure altered the affinity and/or receptor coupling of D2Rs, but not the number of these receptors in heterozygote null Snap25 mutants. These results refine the observations made in the coloboma mouse mutant, a proposed mouse model of ADHD, and illustrate how gene × environmental influences can interact to perturb neural functions that regulate behavior. PMID:23939223

  3. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    SciTech Connect

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-07-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

  4. [76Br]BMK-152, a non-peptide analogue, with high affinity and low non-specific binding for the Corticotropin-Releasing Factor Type 1 Receptor (CRF1 receptor)

    PubMed Central

    Jagoda, Elaine M.; Lang, Lixin; McCullough, Karen; Contoreggi, Carlo; Kim, B. Moon; Ma, Ying; Rice, Kenner C.; Szajek, Lawrence P; Eckelman, William C.; Kiesewetter, Dale O.

    2013-01-01

    Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF1 or CRF2. A PET ligand able to monitor changes in CRF1 receptor occupancy in vivo would aid in understanding the pathophysiology of stress related diseases as well as in the clinical development of non-peptide antagonists with therapeutic value. We have radiolabeled the CRF1 receptor ligand, BMK-152 ([8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine; ClogP= 2.6), at both the 3 and 4 position with [76Br]. Using in vitro autoradiography saturation studies the 4-[76Br]BMK-152 exhibited high affinity binding to both rat (Kd = 0.23 ± 0.07 nM; n=3) and monkey frontal cortex (Kd = 0.31 ± 0.08 nM; n=3) consistent with CRF1 receptor regional distribution whereas with the 3-[76Br]BMK-152, the Kd's could not be determined due to high non-specific binding. In vitro autoradiography competition studies using [125I]Tyr0-o-CRF confirmed that 3-Br-BMK-152 (Ki = 24.4 ± 4.9 nM; n=3) had lower affinity (70 fold) than 4-Br-BMK-152 (Ki = 0.35 ± 0.07 nM; n=3) in monkey frontal cortex and similiar studies using [125I]Sauvagine confirmed CRF1 receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wildtype littermates (WT) showed that the brain uptake of 3-[76Br]BMK/4-[76Br]BMK was increased < 2 fold in KO vs WT indicating that 3-[76Br]BMK-152/4-[76Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[76Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF1 receptor distribution and potential as a PET ligand for in vivo imaging of CRF1 receptors. PMID:21308801

  5. Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

    PubMed Central

    Lee, Terry H. Y.; Mitchell, Ainslie; Liu Lau, Sydney; An, Hongyan; Rajeaskariah, Poornima; Wasinger, Valerie; Raftery, Mark; Bryant, Katherine; Tedla, Nicodemus

    2013-01-01

    The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2–4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn140, Asn281, Asn302, Asn341, and Asn431 sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by β-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions. PMID:24085305

  6. Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue.

    PubMed

    Ma, Xinran; Xu, Lingyan; Mueller, Elisabetta

    2016-03-22

    Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3's expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids. PMID:26957608

  7. Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue

    PubMed Central

    Ma, Xinran; Xu, Lingyan; Mueller, Elisabetta

    2016-01-01

    Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3’s expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids. PMID:26957608

  8. The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

    PubMed

    Ochaion, A; Bar-Yehuda, S; Cohen, S; Barer, F; Patoka, R; Amital, H; Reitblat, T; Reitblat, A; Ophir, J; Konfino, I; Chowers, Y; Ben-Horin, S; Fishman, P

    2009-01-01

    The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation. PMID:19426966

  9. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice.

    PubMed

    Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia; Banala, Ashwini K; Burzynski, Caitlin; Pommier, Elie; Keck, Thomas M; Donthamsetti, Prashant; Javitch, Jonathan A; Rais, Rana; Slusher, Barbara S; Xi, Zheng-Xiong; Newman, Amy Hauck

    2015-08-13

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice. PMID:26203768

  10. Spontaneous expression of a low affinity Fc receptor for IgA (Fc alpha R) on human B cell lines.

    PubMed Central

    Millet, I; Briere, F; Vincent, C; Rousset, F; Andreoni, C; De Vries, J E; Revillard, J P

    1989-01-01

    Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells. PMID:2788048

  11. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.

    PubMed

    Lu, Dan; Shen, Juqun; Vil, Marie D; Zhang, Haifan; Jimenez, Xenia; Bohlen, Peter; Witte, Larry; Zhu, Zhenping

    2003-10-31

    Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides

  12. Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes

    PubMed Central

    Rajapaksha, Harinda; Forbes, Briony E.

    2015-01-01

    The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His4, Tyr15, Thr49, Ile51) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I. PMID:26217307

  13. The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives

    PubMed Central

    Federico, Stephanie; Redenti, Sara; Sturlese, Mattia; Ciancetta, Antonella; Kachler, Sonja; Klotz, Karl-Norbert; Cacciari, Barbara; Moro, Stefano; Spalluto, Giampiero

    2015-01-01

    A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A2B adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA2B AR but they displayed affinity at the hA3 AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA3 AR Ki = 11 nM) and selectivity (A1/A3 and A2A/A3 > 9090; A2B/A3 > 909) at the hA3 AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs. PMID:26625265

  14. Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters

    PubMed Central

    Xu, Rong; Lord, Sarah A.; Peterson, Ryan M.; Fergason-Cantrell, Emily A.; Lever, John R.; Lever, Susan Z.

    2014-01-01

    Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75 nM – 4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20 nM – 30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13 nM – 21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1 Ki = 20.8 nM; σ2 Ki = 16.4 nM) showed over 100-fold higher DAT affinity (Ki = 121 nM) and 6-fold higher SERT affinity (Ki = 128 nM) than the parent SA4503 (DAT Ki = 12650 nM; SERT Ki = 760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions. PMID:25468036

  15. Characterization of radioiodinated TISCH: a high-affinity and selective ligand for mapping CNS D1 dopamine receptor.

    PubMed

    Billings, J J; Kung, M P; Chumpradit, S; Mozley, D; Alavi, A; Kung, H F

    1992-01-01

    In developing CNS D1 dopamine receptor-imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], an analog of SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)-[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(-) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD = 0.21 +/- 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 greater than (+/-)-TISCH greater than (+)-butaclamol = (+/-)-FISCH [7-chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3-benzazepine] much greater than WB4101 = spiperone greater than dopamine, serotonin, (+/-)-propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)-[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)-TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine-123, it may be suitable for noninvasive imaging in humans. PMID:1530770

  16. The potency and efficacy of anticholinergics to inhibit haloperidol-induced catalepsy in rats correlates with their rank order of affinities for the muscarinic receptor subtypes.

    PubMed

    Erosa-Rivero, Helena B; Bata-García, José L; Alvarez-Cervera, Fernando J; Heredia-López, Francisco J; Góngora-Alfaro, José L

    2014-06-01

    Extrapyramidal syndromes (EPS) caused by antipsychotic therapy are currently treated with anticholinergics that lack selectivity for the five muscarinic receptor subtypes. Since these receptors are heterogeneously expressed among the different classes of striatal neurons and their afferents, it can be expected that their simultaneous blockade will cause distinct, sometimes opposed, effects within the striatal circuitry. In order to test the hypothesis that the differential blockade of the muscarinic receptor subtypes would influence their potency and efficacy to prevent EPS, here we tested four anticholinergics with varying order of affinities for the muscarinic receptor subtypes, and compared their dose-response curves to inhibit haloperidol-induced catalepsy in male rats. Drugs were applied into the lateral ventricle 15 min before haloperidol (2 mg/kg, s.c.). Catalepsy was measured in the bar test at 15 min intervals during 5 h. The preferential M1/M4 antagonist pirenzepine (3, 10, 30, 100, and 300 nmol) caused a dose-dependent inhibition of catalepsy intensity: ED50 = 5.6 nmol [95% CI, 3.9-8.1], and latency: ED50 = 5.6 nmol [95% CI, 3.7-8.6]. Pirenzepine had the steepest dose-response curve, producing maximal inhibition (84 ± 5%) at the dose of 10 nmol, while its effect tended to reverse at higher doses (62 ± 11%). The purported M1/M3 antagonist 4-DAMP (30, 100, and 300 nmol) also caused a dose-dependent inhibition of catalepsy intensity: ED50 = 29.5 nmol [95% CI, 7.0 to 123.0], and latency: ED50 = 28.5 nmol [95% CI, 2.2 to 362.0]. However, the curve for 4-DAMP had a less pronounced slope, reaching its maximal effect (63 ± 14%) at the dose of 300 nmol. The M2/M4 antagonist AF-DX 116 (10, 30, and 300 nmol) only caused a partial inhibition of catalepsy (30 ± 11%) at the dose of 30 nmol, but this changed to a non-significant increment (15 ± 10%) at the dose of 100 nmol. The alleged M4 antagonist tropicamide (30, 100, 300, and

  17. Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2*

    PubMed Central

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle; van Agthoven, Jan; Raynal, Bertrand; Hoos, Sylviane; Kragelund, Birthe B.; Kelly, Paul A.; Ducruix, Arnaud; England, Patrick; Goffin, Vincent

    2010-01-01

    We report the first crystal structure of a 1:2 hormone·receptor complex that involves prolactin (PRL) as the ligand, at 3.8-Å resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL·PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 (“three-pin plug”): the conserved glycine 129 of helix α3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL·PRLR2 structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer. PMID:20053995

  18. Quantification of human opiate receptor concentration and affinity using high and low specific activity ( sup 11 C)diprenorphine and positron emission tomography

    SciTech Connect

    Sadzot, B.; Price, J.C.; Mayberg, H.S.; Douglass, K.H.; Dannals, R.F.; Lever, J.R.; Ravert, H.T.; Wilson, A.A.; Wagner, H.N. Jr.; Feldman, M.A. )

    1991-03-01

    (11C)Diprenorphine, a weak partial opiate agonist, and positron emission tomography were used to obtain noninvasive regional estimates of opiate receptor concentration (Bmax) and affinity (Kd) in human brain. Different compartmental models and fitting strategies were compared statistically to establish the most reliable method of parameter estimation. Paired studies were performed in six normal subjects using high (769-5,920 Ci/mmol) and low (27-80 Ci/mmol) specific activity (SA) (11C)diprenorphine. Two subjects were studied a third time using high SA (11C)diprenorphine after a pretreatment with 1-1.5 mg/kg of the opiate antagonist naloxone. After the plasma radioactivity was corrected for metabolites, the brain data were analyzed using a three-compartment model and nonlinear least-squares curve fitting. Linear differential equations were used to describe the high SA (low receptor occupancy) kinetics. The k3/k4 ratio varied from 1.0 +/- 0.2 (occipital cortex) to 8.6 +/- 1.6 (thalamus). Nonlinear differential equations were used to describe the low SA (high receptor occupancy) kinetics and the curve fits provided the konf2 product. The measured free fraction of (11C)diprenorphine in plasma (f1) was 0.30 +/- 0.03, the average K1/k2 ratio from the two naloxone studies was 1.1 +/- 0.2, and the calculated free fraction of (11C)diprenorphine in the brain (f2) was 0.3. Using the paired SA studies, the estimated kinetic parameters, and f2, separate estimates of Bmax and Kd were obtained. Bmax varied from 2.3 +/- 0.5 (occipital cortex) to 20.6 +/- 7.3 (cingulate cortex) nM. The average Kd (eight brain regions) was 0.85 +/- 0.17 nM.

  19. Affinity chromatography of alpha/sub 2/-adrenergic receptors (. cap alpha. /sub 2/AR) from pig cerebral cortex

    SciTech Connect

    Repaske, M.G.; Limbird, L.E.

    1986-03-01

    A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by coupling yohimbinic acid to diaminodipropylamine agarose with 1,3 dicyclohexylcarbodiimide. Unreacted amino groups on the agarose matrix are blocked subsequently by acetylation. One volume of YOH. ag adsorbs 75% of the ..cap alpha../sub 2/AR from 50 volumes of digitonin-solubilized preparation containing 0.2 pmol ..cap alpha../sub 2/AR/mg protein. Digitonin-solubilized preparations are derived from cholate extracts of porcine cerebral cortex containing approx. 0.075 pmol ..cap alpha../sub 2/AR/mg protein. Adsorption of ..cap alpha../sub 2/AR to YOH. ag is selective and thus is blocked by the ..cap alpha..-adrenergic antagonist phentolamine. Adsorbed ..cap alpha../sub 2/AR are eluted with 10 ..mu..M phentolamine (20% yield) after removal of non-related proteins with NaCl gradients. Following hydroxylapatite chromatography to concentrate ..cap alpha..''AR and to remove phentolamine, the ..cap alpha..AR is present at 200-400 pmol/mg protein, assayed using sub-saturating concentrations of (/sup 3/H)-yohimbine. (It is estimated that the specific activity of a homogeneous ..cap alpha../sub 2/AR preparation would be 12,000-16,000 pmol/mg protein.) The availability of large quantities of cortical ..cap alpha../sub 2/AR and a resin easily prepared from commercially-supplied reagents suggests that purification of quantities of ..cap alpha../sub 2/AR sufficient for subsequent biochemical studies is feasible.

  20. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    PubMed

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  1. High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

    PubMed

    Kessler, R M; Ansari, M S; Schmidt, D E; de Paulis, T; Clanton, J A; Innis, R; al-Tikriti, M; Manning, R G; Gillespie, D

    1991-01-01

    Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These

  2. Chemoenzymatic Synthesis and Fcγ Receptor Binding of Homogeneous Glycoforms of Antibody Fc Domain. Presence of a Bisecting Sugar Moiety Enhances the Affinity of Fc to FcγIIIa Receptor

    PubMed Central

    Zou, Guozhang; Ochiai, Hirofumi; Huang, Wei; Yang, Qiang; Li, Cishan; Wang, Lai-Xi

    2011-01-01

    Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on antibody’s effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substratess, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A, nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q), was able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. SPR binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high-affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc

  3. Receptor Surface Models in the Classroom: Introducing Molecular Modeling to Students in a 3-D World

    ERIC Educational Resources Information Center

    Geldenhuys, Werner J.; Hayes, Michael; Van der Schyf, Cornelis J.; Allen, David D.; Malan, Sarel F.

    2007-01-01

    A simple, novel and generally applicable method to demonstrate structure-activity associations of a group of biologically interesting compounds in relation to receptor binding is described. This method is useful for undergraduates and graduate students in medicinal chemistry and computer modeling programs.

  4. Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

    PubMed Central

    Rebollo, A; Silva, A

    1993-01-01

    In order to understand the respective roles of IL-2R alpha and IL-2R beta subunits in transmission of the interleukin-2 (IL-2)-mediated growth signals, we have established two IL-4-dependent murine T-cell clones stably expressing the human IL-2R beta chain and three clones stably expressing the human IL-2R alpha chain. Whereas parental LD8 cells (which express only the murine IL-2R beta chain) do not proliferate in response to IL-2, cell lines stably expressing human IL-2R beta or the chimeric IL-2R alpha beta complex proliferate in response to IL-2. Stably transfected cells expressing the chimeric high-affinity receptor (human IL-2R alpha and murine IL-2R beta) expressed de novo endogenous murine IL-2R alpha when cultured in the presence of IL-2 but not IL-4. Both chimeric and endogenous receptors are functional in response to IL-2, since only addition of both anti-human and anti-murine IL-2R alpha monoclonal antibodies (mAb) inhibited IL-2-induced proliferation. Taken together, our results strongly suggest that human and murine IL-2R beta molecules are different since interaction of IL-2 with human p70 IL-2R is sufficient for transduction of proliferative signals in the absence of p55 IL-2R or, alternatively, that over-expression of the IL-2R beta chain renders cells responsive to IL-2. In addition, IL-2 stimulation of T cells through different forms of IL-2R results in the induction of distinct cellular responses. PMID:8262551

  5. Quantitative Analysis of Human Neonatal Fc Receptor (FcRn) Tissue Expression in Transgenic Mice by Online Peptide Immuno-Affinity LC-HRMS.

    PubMed

    Fan, Yao-Yun; Neubert, Hendrik

    2016-04-19

    Neonatal Fc receptor (FcRn) is the homeostatic receptor responsible for the long half-life of endogenous IgG by protecting it from lysosomal degradation. Understanding systemic FcRn tissue expression is important to predict and design the half-life of therapeutic antibodies and Fc-coupled biotherapeutics. To this end, we measured human FcRn (hFcRn) tissue expression in Tg32, a human FcRn knock-in transgenic mouse model, for which a strong correlation of drug clearance to humans has been demonstrated. Building an hFcRn tissue expression profile in Tg32 was enabled by the development of a tissue preparation procedure composed of bead-based protein extraction and protein precipitation using acetone followed by pellet digestion with trypsin. Digests were then loaded onto an online peptide immuno-affinity flow configuration hyphenated with reversed phase nanoflow chromatography and coupled with high resolution mass spectrometry to quantify hFcRn derived peptides. The workflow allowed bypassing some of the challenges typically associated with membrane protein analysis. We demonstrated acceptable precision and bias for measuring hFcRn in tissue matrices, typically within 20% coefficient of variation and relative error. We also report hFcRn expression in several Tg32 tissues. We anticipate that establishing a quantitative approach for hFcRn in tissues will enable the systematic measurement of hFcRn concentrations to further increase the accuracy of physiologically based pharmacokinetic (PBPK) models for PK prediction of Fc-containing biotherapeutics. This is anticipated to improve the translation of pharmacokinetic data from preclinical model systems to humans. PMID:27012525

  6. Structure-activity studies related to ABT-594, a potent nonopioid analgesic agent: effect of pyridine and azetidine ring substitutions on nicotinic acetylcholine receptor binding affinity and analgesic activity in mice.

    PubMed

    Holladay, M W; Bai, H; Li, Y; Lin, N H; Daanen, J F; Ryther, K B; Wasicak, J T; Kincaid, J F; He, Y; Hettinger, A M; Huang, P; Anderson, D J; Bannon, A W; Buckley, M J; Campbell, J E; Donnelly-Roberts, D L; Gunther, K L; Kim, D J; Kuntzweiler, T A; Sullivan, J P; Decker, M W; Arneric, S P

    1998-10-01

    Analogs of A-98593 (1) and its enantiomer ABT-594 (2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays. PMID:9873625

  7. Prostaglandin A2 enhances cellular insulin sensitivity via a mechanism that involves the orphan nuclear receptor NR4A3.

    PubMed

    Zhu, X; Walton, R G; Tian, L; Luo, N; Ho, S-R; Fu, Y; Garvey, W T

    2013-03-01

    We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin's ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3. PMID:23104421

  8. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

    PubMed Central

    Schloss, P; Hermans-Borgmeyer, I; Betz, H; Gundelfinger, E D

    1988-01-01

    The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system. PMID:3141150

  9. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    PubMed Central

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  10. High-affinity NGF receptor in the rat spinal cord during acute and chronic phases of experimental autoimmune encephalomyelitis: a possible functional significance.

    PubMed

    Oderfeld-Nowak, B; Zaremba, M; Lipkowski, A W; Kwiatkowska-Patzer, B; Triaca, V; Aloe, L

    2003-03-01

    The biological effects of Nerve Growth Factor (NGF) are primarily mediated via its high affinity receptor-TrkA. In the present study, we examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of TrkA in neuronal and non-neuronal cells of the spinal cord of Lewis rats during the acute (14 days postimmunization) and chronic (12 months postimmunization) phases of the disease. In the normal spinal cord, both of mature and aged rats, we found TrkA immunoreaction (TrkA-IR) in the motoneurons of the Rexed lamina IX and in both oligo- and astroglia cells. In the acute phase of the disease, we found a reduction of TrkA immunoreactivity in motoneurons and its up-regulation in oligodendroglia, mainly in the white matter. We also confirmed our previous findings concerning the up-regulation of TrkA-IR in astroglia. Both neuronal and non-neuronal changes of TrkA immunoreactivity had a transient character: they were not seen in the chronic phase of the disease. Our results suggest that both neuronal and glial TrkA expression changes depend on inflammation. Moreover, our data indicate that, during the acute phase of EAE, the glial cells become more receptive to NGF, pointing to glia as an important target for pharmacological manipulations, particularly for exogenously administered NGF. PMID:12825322

  11. Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

    PubMed Central

    2014-01-01

    Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. Results Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNβ in the RAW264.7 challenged with LPS. Conclusion Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action. PMID:24602493

  12. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    PubMed

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β. PMID:26940508

  13. A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

    PubMed Central

    Haridan, Umi Shakina; Mokhtar, Umairah; Machado, Lee R.; Abdul Aziz, Abu Thalhah; Shueb, Rafidah Hanim; Zaid, Masliza; Sim, Benedict; Mustafa, Mahiran; Nik Yusof, Nik Khairudin; Lee, Christopher K. C.; Abu Bakar, Suhaili; AbuBakar, Sazaly; Hollox, Edward J.; Boon Peng, Hoh

    2015-01-01

    The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs. PMID:25594501

  14. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    SciTech Connect

    Shabanpoor, Fazel; Bathgate, Ross A.D.; Belgi, Alessia; Chan, Linda J.; Nair, Vinojini B.; Wade, John D.; Hossain, Mohammed Akhter

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. Black-Right-Pointing-Pointer The choice of a site for incorporation of a chelator is critical. Black-Right-Pointing-Pointer The labeled peptide retains full activity at the RXFP1 receptor. Black-Right-Pointing-Pointer It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to

  15. Genomic organization, promoter analysis, and chromosomal localization of the gene for the mouse glial high-affinity glutamate transporter Slc1a3

    SciTech Connect

    Hagiwara, Tatsuya; Tanaka, Kohichi; Maeno-Hikichi, Yuka

    1996-05-01

    The mouse gene encoding glial high-affinity, Na -dependent glutamate transporter Slcla3 (GluT-1/GLAST) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the mouse genome and comprised 10 exons spanning more than 56 kilobases. The transcription initiation sites were mapped to positions 503, which is the first transcriptional point (defined as +1), 128 (+376), and 64 (+440) basepairs upstream of the 3{prime}-end of exon 1 by primer extension. The 5{prime}-flanking region of the mouse GluT-1 gene had a typical CCAAT box and a GC box but lacked at TATA box. These features of the promoter region were characteristic of housekeeping genes. The fusion plasmids containing approximately 4 kb of the 5{prime}-flanking region (-3830 to +450) and the firefly luciferase gene induced a significant luciferase activity when transfected into COS-1 cells. Distal deletion of the 5{prime}-flanking region, leaving 619 bp (-169 to +450), resulted in a marked decrease in luciferase activity in COS-1 cells, suggesting that a CCAAT box, which was positioned at -200, is necessary for the expression of this gene. In situ hybridization localized this gene. In situ hybridization localized this gene to mouse chromosome 15A2. These structural features will lead to a better understanding of the regulatory mechanism of the expression of the GluT-1 gene by ischemia and will also provide a basis for future evolutionary comparisons with other neurotransmitter transporters. 40 refs., 6 figs., 1 tab.

  16. The A3 adenosine receptor agonist CP-532,903 protects against myocardial ischemia/reperfusion injury via the sarcolemmal ATP sensitive potassium channel

    PubMed Central

    Wan, Tina C.; Ge, Zhi-Dong; Tampo, Akihito; Mio, Yasushi; Bienengraeber, Martin W.; Tracey, W. Ross; Gross, Garrett J.; Kwok, Wai-Meng; Auchampach, John A.

    2008-01-01

    We examined the cardioprotective profile of the new A3 adenosine receptor (AR) agonist CP-532,903 in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using HEK 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A3ARs (ki = 9.0±2.5 nM) and with high selectivity versus mouse A1AR (100-fold) and A2AARs (1,000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 µg/kg of CP-532,903 reduced infarct size from 59.2 ± 2.1% of the risk region in vehicle-treated mice to 42.5 ± 2.3% and 39.0 ± 2.9%, respectively. Similarly, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration-dependently improved recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion, including developed pressure and ±dP/dt. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A3AR gene (A3KO mice). In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by 3 brief ischemia/reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATP-sensitive potassium (KATP) channel. Whole-cell patch clamp recordings provided evidence that the A3AR is functionally coupled to the sarcolemmal KATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A3AR that protects against ischemia/reperfusion injury by activating sarcolemmal KATP channels. PMID:17906066

  17. Novel Fluorescent Antagonist as a Molecular Probe in A3 Adenosine Receptor Binding Assays Using Flow Cytometry

    PubMed Central

    Kozma, Eszter; Kumar, T. Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization. PMID:22402302

  18. Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species.

    PubMed

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y; Peterson-Yantorno, Kim; Stone, Richard A; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A; Civan, Mortimer M

    2010-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  19. Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species

    PubMed Central

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y.; Peterson-Yantorno, Kim; Stone, Richard A.; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A.; Civan, Mortimer M.

    2009-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 minutes. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  20. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that (/sup 3/H)dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    SciTech Connect

    Leff, S.E.; Creese, I.

    1985-02-01

    The interactions of dopaminergic agonists and antagonists with /sup 3/H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of (/sup 3/H)dopamine and (/sup 3/H)apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/(/sup 3/H)dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific (/sup 3/H)dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and (/sup 3/H)flupentixol-binding activities. The affinities of agonists to inhibit D3 specific (/sup 3/H)dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/(/sup 3/H)flupentixol competition curves. Both D3 specific (/sup 3/H) dopamine binding and the high affinity agonist-binding component of dopamine/(/sup 3/H)flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.

  1. The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

    PubMed

    Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

    2008-08-15

    The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis. PMID:18602896

  2. The A3 Adenosine Receptor Agonist CF502 Inhibits the PI3K, PKB/Akt and NF-κB Signaling Pathway in Synoviocytes from Rheumatoid Arthritis Patients and in Adjuvant Induced Arthritis Rats

    PubMed Central

    Ochaion, A.; Bar-Yehuda, S.; Cohen, S.; Amital, H.; Jacobson, K.A.; Joshi, B.V.; Gao, Z.G.; Barer, F.; Patoka, R.; Del Valle, L.; Perez-Liz, G.; Fishman, P.

    2009-01-01

    The A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant and collagen induced arthritis. In this study we present a novel A3AR agonist, CF502, with high affinity and selectivity at the human A3AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of Adjuvant Induced Arthritis (AIA) in a rat experimental model when given orally at a low dose (100 μg/kg). As is typical of other G-protein coupled receptors, the A3AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of Protein Kinase B/Akt (PKB/Akt), IκB kinase (IKK), (I kappa B) IκB, NF-κB and tumor necrosis factor-alpha (TNF-α) took place. In addition, the expression levels of Glycogen synthase kinase-3 beta (GSK-3β), β-catenin, and Poly (ADP-ribose) polymerase (PARP), known to control the level and activity of NF-κB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A3AR agonists for the treatment of rheumatoid arthritis. PMID:18602896

  3. Development of p-benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) derivatives and affinity photolabeling of the bovine NPR-A receptor.

    PubMed

    Coupal, M; De Léan, A; McNicoll, N; Fournier, A

    1999-04-29

    Two Nalpha-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd = 20 pM) with Kd of 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2 or Leu18 can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling. PMID:10222239

  4. A [3]rotaxane with two porphyrinic plates acting as an adaptable receptor.

    PubMed

    Frey, Julien; Tock, Christian; Collin, Jean-Paul; Heitz, Valérie; Sauvage, Jean-Pierre

    2008-04-01

    Following a multistep procedure, the copper(I)-templated strategy allowed preparation of a multifunctional [3]rotaxane. The dumbbell consists of a central two-bidentate chelate unit and two terminal stoppers. The two rings threaded on the rotaxane axis consist each of a 1,10-phenanthroline-incorporating macrocycle, rigidly connected to an appended zinc-complexed porphyrin. The copper(I) template can be removed, affording a free rotaxane whose two rings can glide freely along the axis and spin around it. The dumbbell being very long (approximately 85 A in its extended conformation from one stopper to the other), the porphyrin-porphyrin distance can be varied over a wide range. The two porphyrinic plates constitute the key elements of a receptor able to complex various guests between the plates. The ability of the threaded rings to move freely makes the host perfectly adjustable, allowing capture of geometrically very different guests. The copper(I)-complexed rotaxane also acts as an efficient receptor, although its adaptability is obviously more limited than that of its free rotaxane counterpart. PMID:18338892

  5. Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients.

    PubMed

    Kosugi, S; Ban, T; Akamizu, T; Kohn, L D

    1991-10-31

    Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site. PMID:1719963

  6. Pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one as a new scaffold to develop potent and selective human A3 adenosine receptor antagonists. Synthesis, pharmacological evaluation, and ligand-receptor modeling studies.

    PubMed

    Colotta, Vittoria; Lenzi, Ombretta; Catarzi, Daniela; Varano, Flavia; Filacchioni, Guido; Martini, Claudia; Trincavelli, Letizia; Ciampi, Osele; Pugliese, Anna Maria; Traini, Chiara; Pedata, Felicita; Morizzo, Erika; Moro, Stefano

    2009-04-23

    The paper describes a new class of human (h) A(3) adenosine receptor antagonists, the 2-arylpyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one derivatives (PTP), either 4-oxo (1-6, series A) or 4-amino-substituted (7-20, series B). In both series A and B, substituents able to act as hydrogen bond acceptors (OMe, OH, F, COOEt) were inserted on the 2-phenyl ring. In series B, cycloalkyl and acyl residues were introduced on the 4-amino group. Some of the new derivatives showed high hA(3) AR affinities (K(i) < 50 nM) and selectivities vs both hA(1) and hA(2A) receptors. The selected 4-benzoylamino-2-(4-methoxyphenyl)pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one (18), tested in an in vitro rat model of cerebral ischemia, proved to be effective in preventing the failure of synaptic activity induced by oxygen and glucose deprivation in the hippocampus. Molecular docking of this new class of hA(3) AR antagonists was carried out to depict their hypothetical binding mode to our refined model of hA(3) receptor. PMID:19301821

  7. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    PubMed Central

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  8. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

    PubMed

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  9. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    PubMed

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema. PMID:27150147

  10. Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

    PubMed Central

    Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

    2014-01-01

    A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

  11. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone.

    PubMed

    Shabanpoor, Fazel; Bathgate, Ross A D; Belgi, Alessia; Chan, Linda J; Nair, Vinojini B; Wade, John D; Hossain, Mohammed Akhter

    2012-04-01

    Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide. PMID:22425984

  12. Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS

    PubMed Central

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G.; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J.; Filizola, Marta; Springer, Timothy A.; Coller, Barry S.

    2012-01-01

    An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent. PMID:22422993

  13. Mapping the binding pocket of a novel, high-affinity, slow dissociating tachykinin NK3 receptor antagonist: biochemical and electrophysiological characterization.

    PubMed

    Malherbe, Pari; Knoflach, Frédéric; Marcuz, Anne; Bohnert, Claudia; Weber, Michael; Knust, Henner; Ratni, Hasane; Spooren, Will; Ballard, Theresa M; Bissantz, Caterina

    2014-11-01

    The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3. PMID:25107588

  14. CF102 an A3 Adenosine Receptor Agonist Mediates Anti-Tumor and Anti-Inflammatory Effects in the Liver

    PubMed Central

    COHEN, S.; STEMMER, S.M.; ZOZULYA, G.; OCHAION, A.; PATOKA, R.; BARER, F.; BAR-YEHUDA, S.; RATH-WOLFSON, L.; JACOBSON, K.A.; FISHMAN, P.

    2012-01-01

    The Gi protein-associated A3 adenosine receptor (A3AR) is a member of the adenosine receptor family. Selective agonists at the A3AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 μg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  15. CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver.

    PubMed

    Cohen, S; Stemmer, S M; Zozulya, G; Ochaion, A; Patoka, R; Barer, F; Bar-Yehuda, S; Rath-Wolfson, L; Jacobson, K A; Fishman, P

    2011-09-01

    The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  16. Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

    SciTech Connect

    Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.; Katamreddy, Subba R.; Navas III, Frank; Miller, Aaron B.; Williams, Shawn P.; Gray, David W.; Orband-Miller, Lisa A.; Shearin, Jean; Heyer, Dennis

    2009-06-24

    A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from microm