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issn:0026-895
1.  Selective Potentiation of Paclitaxel (Taxol)-Induced Cell Death by Mitogen-Activated Protein Kinase Kinase Inhibition in Human Cancer Cell Lines 
Molecular pharmacology  2001;60(2):290-301.
Activation of the mitogen-activated protein kinase (MAPK) pathway in HeLa and Chinese hamster ovary cells after treatment with paclitaxel (Taxol) and other microtubule interacting agents has been investigated. Using a trans-reporting system, the phosphorylation of the nuclear transcription factors Elk-1 and c-jun was measured. Concentration- and time-dependent activation of the Elk-1 pathway, mediated primarily by the extracellular signal-regulated kinase (ERK) component of the MAPK family, was observed. Inactive drug analogs and other cytotoxic compounds that do not target microtubules failed to induce similar levels of activation, thereby indicating that an interaction between these drugs and the microtubule is essential for the activation of MAPKs. Evaluation of the endogenous levels of MAPK expression revealed cell-dependent expression of the ERK, c-jun N-terminal kinase, and p38 pathways. In the case of HeLa cells, time-dependent activation of ERK coincided with increased poly(ADP-ribose) polymerase (PARP) cleavage, phosphatidylserine externalization, and increased accumulation of cells in G2M. In both cell lines, inhibition of ERK activity potentiated paclitaxel-induced PARP cleavage and phosphatidylserine externalization, suggesting that ERK activity coincided with, but did not mediate, the cytotoxic effects of paclitaxel. We evaluated the nature of the interaction between paclitaxel and the MAPK kinase inhibitor U0126 in three cell lines, on the basis of a potential chemotherapeutic advantage of paclitaxel plus ERK inhibition. Our data confirmed additivity in those cells lines that undergo paclitaxel-induced ERK activation, and antagonism in cells with low ERK activity, suggesting that in tumors with high ERK activity, there may be an application for this strategy in therapy.
PMCID: PMC4039042  PMID: 11455016
2.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
PMCID: PMC3953614  PMID: 11641434
3.  Selective Allosteric Enhancement of Agonist Binding and Function at Human A3 Adenosine Receptors by a Series of Imidazoquinoline Derivatives 
Molecular pharmacology  2002;62(1):81-89.
We have identified a series of 1H-imidazo-[4,5-c]quinolines as selective allosteric enhancers of human A3 adenosine receptors. Several of these compounds potentiated both the potency and maximal efficacy of agonist-induced responses and selectively decreased the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors. There was no effect on the dissociation of the antagonist [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) from the A3 receptors, as well as [3H]N6-[(R)-phenylisopropy-l]adenosine from rat brain A1 receptors and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoad-enosine from rat striatal A2A receptors, suggesting the selective enhancement of agonist binding at A3 receptors. The analogs were tested as antagonists of competitive binding at human A3 receptors, and Ki values ranging from 120 nM to 101 μM were observed; as for many allosteric modulators of G protein-coupled receptors, an orthosteric effect was also present. The most promising leads from the present set of analogs seem to be the 2-cyclopentyl-1H-imidazo[4,5-c]quinoline derivatives, of which the 4-phenylamino analog DU124183 had the most favorable degree of allosteric modulation versus receptor antagonism. The inhibition of forskolin-stimulated cyclic AMP accumulation in intact cells that express human A3 receptors was employed as a functional index of A3 receptor activation. The enhancer DU124183 caused a marked leftward shift of the concentration-response curve of the A3 receptor agonists in the presence of antagonist and, surprisingly, a potentiation of the maximum agonist efficacy by approximately 30%. Thus, we have identified a novel structural lead for developing allosteric enhancers of A3 adenosine receptors; such enhancers may be useful for treating brain ischemia and other hypoxic conditions.
PMCID: PMC3953617  PMID: 12065758
4.  An Opioid Agonist that Does Not Induce μ-Opioid Receptor—Arrestin Interactions or Receptor Internalization 
Molecular pharmacology  2006;71(2):549-557.
G protein-coupled receptor desensitization and trafficking are important regulators of opioid receptor signaling that can dictate overall drug responsiveness in vivo. Furthermore, different μ-opioid receptor (μOR) ligands can lead to varying degrees of receptor regulation, presumably because of distinct structural conformations conferred by agonist binding. For example, morphine binding produces a μOR with low affinity for β-arrestin proteins and limited receptor internalization, whereas enkephalin analogs promote robust trafficking of both β-arrestins and the receptors. Here, we evaluate μOR trafficking in response to activation by a novel μ-selective agonist derived from the naturally occurring plant product, salvinorin A. It is interesting that this compound, termed herkinorin, does not promote the recruitment of β-arrestin-2 to the μOR and does not lead to receptor internalization. Moreover, whereas G protein-coupled receptor kinase overexpression can promote morphine-induced β-arrestin interactions and μOR internalization, such manipulations do not promote herkinorin-induced trafficking. Studies in mice have shown that β-arrestin-2 plays an important role in the development of morphine-induced tolerance, constipation, and respiratory depression. Therefore, drugs that can activate the receptor without recruiting the arrestins may be a promising step in the development of opiate analgesics that distinguish between agonist activity and receptor regulation and may ultimately lead to therapeutics designed to provide pain relief without the adverse side effects normally associated with the opiate narcotics.
doi:10.1124/mol.106.028258
PMCID: PMC3926195  PMID: 17090705
5.  Human methyl purine DNA glycosylase and DNA polymerase ß expression collectively predict sensitivity to temozolomide 
Molecular pharmacology  2008;74(2):505-516.
Over-expression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and shRNA-mediated loss of Pol ß expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol ß or human Pol ß with an inactivating mutation specific to the polymerase active site yet functional for 5′dRP lyase activity. These genetic and cellular studies uniquely demonstrate that over-expression of MPG causes an imbalance in BER leading to an accumulation of cytotoxic 5′dRP lesions and that the 5′dRP lyase activity of Pol ß is required to restore resistance to temozolomide. These results imply that Pol ß dependent 5′dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-MeG and N3-MeA. Further, we find that 5′dRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by GFP-LC3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide.
doi:10.1124/mol.108.045112
PMCID: PMC3909956  PMID: 18477668
6.  Probing the Binding Sites and Mechanisms of Action of Two Human Ether-a-go-go-Related Gene Channel Activators, 1,3-bis-(2-Hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and 2-[2-(3,4-Dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD307243)s 
Molecular pharmacology  2008;73(6):1709-1721.
We studied the mechanisms and sites of activator actions of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid [PD307243 (PD)] and 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea [NS1643 (NS)] on the human ether-ago-go-related gene (hERG) channel expressed in oocytes and COS-7 cells. PD and NS affected hERG in a concentration-dependent manner, reaching a maximal increase in current amplitude by 100% and ≥300% (1-s test pulse to 0 mV), with apparent Kd values of 3 and 20 μM, respectively. Both drugs slowed hERG inactivation. NS additionally shifted the activation curve in the negative direction, accelerated activation, and slowed deactivation. Kinetic model simulations suggested that the activator effects of PD and NS could be largely accounted for by their effects on the hERG gating kinetics. Both drugs worked from outside the cell membrane but their binding sites seemed to be distinctly different. Perturbing the conformation of outer vestibule/external pore entrance (by cysteine substitution at high-impact positions or cysteine side chain modification at intermediate-impact positions) prevented the activator effect of NS but not that of PD. Furthermore, the peptide toxin BeKm-1, which bound to the outer mouth of the hERG channel, suppressed NS effect but potentiated PD effect. We propose that NS is a “gating-modifier”: it binds to the outer vestibule/ pore entrance of hERG and increases current amplitudes by promoting channel activation while retarding inactivation. The activator effect of PD was prevented by external quaternary ammonium cations or dofetilide, which approached the hERG selectivity filter from opposite sides of the membrane and depleted K+ ions in the selectivity filter. We suggest that PD may work as a “pore-modifier” of the hERG channel.
doi:10.1124/mol.108.045591
PMCID: PMC3888322  PMID: 18372399
7.  Expression of Human NAD(P)H:Quinone Oxidoreductase (DT-Diaphorase) in Chinese Hamster Ovary Cells: Effect on the Toxicity of Antitumor Quinones 
Molecular pharmacology  1996;50(4):728-735.
SUMMARY
Previous studies have indicated that NAD(P)H:quinone oxidoreductase [DT-diaphorase (NQO1)] plays an important role in the bioreductive activation of quinone-containing antitumor agents. Although these studies demonstrated that purified NQO1 can reduce these compounds in vitro, the importance NQO1 in the intracellular activation of quinone-containing antitumor agents remains controversial. In our study, we transfected human NQO1 into Chinese hamster ovary cells that do not normally express NQO1 activity and obtained stable clones that expressed NQO1 activity of 19–3527 nmol of 2,6-dichlorophenolindophenol reduced/min/mg of protein. The level of NQO1 expression correlated with an increased killing by streptonigrin, EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propenol), and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone, but mitomycin C sensitivity was independent of this activity. NQO1 expression also led to a slight decrease in the sensitivity of cells to menadione. Our data demonstrate that compounds that are efficient substrates for NQO1 in vitro are also bioactivated in cultured mammalian cells when they are transfected with human NQO1. These results are consistent with the relative abilities of mitomycin C, streptonigrin, EO9, and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone to serve as substrates for bioreduction by human NQO1, and show that NQO1 levels are not necessarily predictive in terms of sensitivity to mitomycin C.
PMCID: PMC3883621  PMID: 8863816
8.  Spatial Approximation between Secretin Residue Five and the Third Extracellular Loop of Its Receptor Provides New Insight into the Molecular Basis of Natural Agonist Binding 
Molecular pharmacology  2008;74(2):10.1124/mol.108.047209.
The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.
doi:10.1124/mol.108.047209
PMCID: PMC3879803  PMID: 18467541
9.  THE COACTIVATOR NCOA6 MEDIATES THE SYNERGISTIC ACTIVATION OF HUMAN CYTOCHROME P-450 2C9 BY THE CONSTITUTIVE ANDROSTANE RECEPTOR AND HEPATIC NUCLEAR FACTOR-4α 
Molecular pharmacology  2008;74(3):10.1124/mol.108.048983.
Nuclear receptor coactivator 6 (NCOA6) also known as PRIP/RAP250/ASC-2 anchors a steady state complex of cofactors and function as a transcriptional coactivator for certain nuclear receptors. This is the first study to identify NCOA6 as an HNF4α interacting protein. CYP2C9 is an important enzyme which metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have previously shown that CAR (a xenobiotic sensing receptor) upregulates the CYP2C9 promoter through binding to a distal site, while HNF4α trancriptionally upregulates CYP2C9 via proximal sites. We demonstrate ligand enhanced synergistic cross talk between CAR and HNF4α. We now identify NCOA6 as crucial to the underlying mechanism of this crosstalk. NCOA6 was identified as an HNF4α interacting protein in this study using a yeast two hybrid screen and GST-pulldown assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4α in HepG2 cells. While the interaction of NCOA6 with CAR is specifically through the first LXXLL motif of NCOA6, both LXXLL motifs are involved in its interaction with HNF4α. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4α. ChIP analysis revealed that NCOA6 can pull down both the proximal HNF4α and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4α.
doi:10.1124/mol.108.048983
PMCID: PMC3815599  PMID: 18552123
10.  Conformational Flexibility of Helix VI is Essential for Substrate Permeation of the Human Apical Sodium-dependent Bile Acid Transporter (ASBT) 
Molecular pharmacology  2007;73(2):10.1124/mol.107.041640.
The present study characterizes the methanethiosulfonate (MTS) inhibition profiles of 26 consecutive cysteine-substituted mutants comprising transmembrane (TM) helix 6 of the human apical Na+-dependent bile acid transporter (hASBT, SLC10A2). TM6 is linked exofacially to TM7 via extracellular loop (EL) 3. TM7 was previously identified as lining part of the substrate permeation path (Hussainzada et al. Mol Pharmacol 70:1565, 2006). Most TM6 cysteine replacements were well-tolerated, except for five residues with either severely hampered (I229C, G249C) or abolished (P234C, G237C, G241C) activity. Disruption of protein synthesis or folding and stability may account for lack of activity for mutant P234C. Subsequent Pro234 amino acid replacement reveals its participation in both structural and functional aspects of the transport cycle. Application of polar MTS reagents (1 mM) significantly inhibited activity of six mutants (V235C, S239C, F242C, R246C, A248C, Y253C), for which rates of modification were almost fully reversed (except Y253C) upon inclusion of bile acid substrates or removal of Na+ from the MTS pre-incubation medium. Activity assessments at equilibrative [Na+] revealed numerous Na+ sensitive residues, suggesting their proximity in or around Na+ interaction sites. In silico modeling reveals the intimate and potentially cooperative orientation of MTS-accessible TM6 residues toward functionally important TM7 amino acids, substantiating TM6 participation during the transport cycle. We conclude a functional requirement for helical flexibility imparted by Pro234, Gly237 and Gly241, likely forming a “conformational switch” requisite for substrate turnover; meanwhile, MTS-accessible residues, which line a helical face spatially distinct from this switch, may participate during substrate permeation.
doi:10.1124/mol.107.041640
PMCID: PMC3812437  PMID: 17971420
11.  Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-directed Mutagenesis and Affinity Inhibition 
Molecular pharmacology  2008;73(4):10.1124/mol.107.043190.
Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-3H-methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only Lys411Ala RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-3H-MTX, wild type (wt) RFC was labeled; for Lys411Ala RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only Lys411Glu RFC showed substantially decreased transport. Nine classical diamino furo[2,3-d]pyrimidine antifolates with unsubstituted α- and γ-carboxylates (1), hydrogen- or methyl-substituted α- (2, 3) or γ- (4, 5) carboxylates, or substitutions of both α- and γ-carboxylates (6, 7, 8, 9) were used to inhibit 3H-MTX transport with RFC-null K562 cells expressing wt and Lys411Ala RFCs. For wt and Lys411Ala RFCs, inhibitory potencies were in the order 4>5>1>3>2; 6-9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2>1>4; inhibition was abolished for Lys411Ala RFC. These results suggest that Lys411 participates in substrate binding via an ionic association with the substrate γ-carboxylate, however, this is not essential for transport. An unmodified α-carboxylate is required for high affinity substrate binding to RFC, whereas the γ-carboxyl is not essential.
doi:10.1124/mol.107.043190
PMCID: PMC3806200  PMID: 18182479
12.  Pharmacological Characterization of Membrane-Expressed Human Trace Amine-Associated Receptor 1 (TAAR1) by a Bioluminescence Resonance Energy Transfer cAMP Biosensor 
Molecular pharmacology  2008;74(3):585-594.
Trace amines are neurotransmitters whose role in regulating invertebrate physiology has been appreciated for many decades. Recent studies indicate that trace amines may also play a role in mammalian physiology by binding to a novel family of G protein-coupled receptors (GPCRs) that are found throughout the central nervous system. A major obstacle impeding the careful pharmacological characterization of trace amine associated receptors (TAARs) is their extremely poor membrane expression in model cell systems, and a molecular basis for this phenomenon has not been determined. In the present study, we show that the addition of an asparagine-linked glycosylation site to the N terminus of the human trace amine associated receptor 1 (TAAR1) is sufficient to enable its plasma membrane expression, and thus its pharmacological characterization with a novel cAMP EPAC (exchange protein directly activated by cAMP) protein based bioluminescence resonance energy transfer (BRET) biosensor. We applied this novel cAMP BRET biosensor to evaluate the activity of putative TAAR1 ligands. This study represents the first comprehensive investigation of the membrane-expressed human TAAR1 pharmacology. Our strategy to express TAARs and to identify their ligands using a cAMP BRET assay could provide a foundation for characterizing the functional role of trace amines in vivo and suggests a strategy to apply to groups of poorly expressing GPCRs that have remained difficult to investigate in model systems.
doi:10.1124/mol.108.048884
PMCID: PMC3766527  PMID: 18524885
13.  Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element 
Molecular pharmacology  1998;54(6):1113-1117.
The pregnenolone X receptor (PXR), a new member of the nuclear hormone receptor superfamily, was recently demonstrated to mediate glucocorticoid agonist and antagonist activation of a hormone response element spaced by three nucleotides (DR-3) within the rat CYP3A23 promoter. Because many other steroids and xenobiotics can up-regulate CYP3A23 expression, we determined whether some of these other regulators used PXR to activate the CYP3A23 DR-3. Transient cotransfection of LLC-PK1 cells with (CYP3A23)2-tk-CAT and mouse PXR demonstrated that the organochlorine pesticides transnonachlor and chlordane and the nonplanar polychlorinated biphenyls (PCBs) each induced the CYP3A23 DR-3 element, and this activation required PXR. Additionally, this study found that PXR is activated to induce (CYP3A23)2-tk-CAT by antihormones of several steroid classes including the antimineralocorticoid spironolactone and the antiandrogen cyproterone acetate. These studies reveal that PXR is involved in the induction of CYP3A23 by pharmacologically and structurally distinct steroids and xenobiotics. Moreover, PXR-mediated PCB activation of the (CYP3A23)2-tk-CAT may serve as a rapid assay for effects of nonplanar PCBs.
PMCID: PMC3662300  PMID: 9855641
14.  Regulation of Nerve Growth Factor Release by Nitric Oxide through Cyclic GMP Pathway in Cortical Glial Cells 
Molecular pharmacology  1999;56(2):339-347.
In the present study, we found that S-nitroso-N-acetyl-dl-penicillamine, a spontaneous nitric oxide (NO) generator, dose-dependently inhibited basal nerve growth factor (NGF) release from mixed glial cells. To elucidate the function of endogenous NO in the regulation of NGF release, the mixed glial cells were stimulated with lipopolysaccharide (LPS) or LPS plus interfer-on-γ (IFNγ). The results showed that LPS alone induced NGF release and moderate NO production. However, costimulation with LPS plus IFNγ greatly enhanced NO production but significantly suppressed LPS-induced NGF release. When NG-monomethyl-l-arginine, an NOS inhibitor, was added to the culture, the suppression of NGF release by IFNγ was significantly reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated S-nitroso-N-acetyl-dl-penicillamine was also able to inhibit the LPS-induced NGF mRNA expression. To understand the different contributions of astroglia and microglia to this phenomenon, both cell types were purified. We found purified astroglia produced high amounts of NGF but low amounts of NO. However, purified microglia produced a large amount of NO but very low amounts of NGF after stimulation with LPS or LPS plus IFNγ. Our data also indicated the second messenger cyclic GMP, but not cyclic AMP, was able to inhibit basal NGF release. In vivo experiments confirmed that NGF protein level was significantly enhanced in rats treated with l-Nω-nitro-arginine methyl ester and in endothelial NO synthase mutant mice. Taken together, we conclude NO derived mainly from microglia down-regulates NGF release from astroglia at the transcriptional level by stimulating cyclic GMP pathway.
PMCID: PMC3659795  PMID: 10419553
15.  Identification of a Functionally Relevant Cannabinoid Receptor on Mouse Spleen Cells that Is Involved in Cannabinoid-Mediated Immune Modulation 
Molecular pharmacology  1992;42(5):736-742.
SUMMARY
Extensive behavioral and biochemical characterization of cannabinoid-mediated effects on the central nervous system has revealed at least three lines of evidence supporting the role of a putative guanine nucleotide-binding protein-coupled cannabinoid receptor for cannabimimetic effects, (i) stereoselectivity, (ii) inhibition of the adenylate cyclase/cAMP second messenger system, and (iii) radioligand-binding studies with the synthetic cannabinoid [3H]CP-55,940 indicating a high degree of specific binding to brain tissue preparations. Based on recent findings from our laboratory demonstrating that Δ9-tetrahydrocannabinol markedly inhibited forskolin-stimulated cAMP accumulation in mouse spleen cells, the presence of a guanine nucleotide-binding protein-coupled cannabinoid receptor associated with mouse spleen cells and its functional role in immune modulation were investigated. In the present studies, stereoselective immune modulation was observed with the synthetic bicyclic cannabinoid (−)-CP-55,940 versus (+) CP-56,667 and with 11-OH-Δ8-tetrahydrocannabinol-dimethylheptyl, (−)-HU-210 versus (+)-HU-211. In both cases, the (−)-enantiomer demonstrated greater immunoinhibitory potency than the (+)-isomer, as measured by the in vitro sheep red blood cell antibody-forming cell response. Radioligand binding studies produced a saturation isotherm exhibiting approximately 45–65% specific binding to mouse spleen cells. Scatchard analysis demonstrated a single binding site on spleen cells, possessing a Kd of 910 pm and a Bmax of approximately 1000 receptors/spleen cell. RNA polymerase chain reaction of isolated splenic RNA using specific primers for the cannabinoid receptor resulted in the amplification of a 854-kilobase predicted product that hybridized with cannabinoid receptor cDNA, demonstrating the presence of cannabinoid receptor mRNA in mouse spleen. Together, these findings strongly support the role of a cannabinoid receptor in immune modulation by cannabimimetic agents.
PMCID: PMC3657723  PMID: 1279376
16.  [No title available] 
PMCID: PMC3640555  PMID: 18198283
17.  High Affinity Acylating Antagonists for the A1 Adenosine Receptor: Identification of Binding Subunit 
Molecular pharmacology  1988;34(6):724-728.
SUMMARY
Two isomenc isothiocyanate derivatives of the A1 adenosine receptor antagonist xanthine amine cogener (XAC) have been synthesized and found to be potent affinity labels (irreversibly bound ligands) for A1 adenosine receptors. The interaction of m- and p-isomers of 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl(2-aminoethylaminocarbonyl(4-methyloxy(phenyl)))))-xanthine (DITC-XAC) with rat brain A1 receptors is of high affinity (EC50 = 27 and 52 nm, respectively) as determined by radioligand competition curves. These compounds reduced the number of A1 receptors (>90% at 500 nm m-DITC-XAC) in brain membranes, without any change in the affinity of the remaining receptors for [125I]N6-2-(4-aminophenyl)ethyladenosine. Prior reaction of the isothiocyanate moiety with ethylenediamine did not alter the affinity of the XAC derivative for the A1 receptor but eliminated its ability to covalently incorporate into the receptor. Incubation of brain membranes with radiolabeled p- and m-DITC-XAC results in the specific labeling of a Mr 38,000 peptide. This labeling can be blocked with both an A1 adenosine receptor-specific agonist and an antagonist. This specific protein has the same molecular weight as the protein labeled with A1-selective photoaffinity probes. The much higher efficiency of incorporation of these affinity probes compared with photoaffinity probes should make them extremely useful for structural studies of A1 adenosine receptors.
PMCID: PMC3557832  PMID: 3200248
18.  Computer-Aided Discovery, Validation, and Mechanistic Characterization of Novel Neolignan Activators of Peroxisome Proliferator-Activated Receptor γ 
Molecular pharmacology  2010;77(4):559-566.
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPARγ agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARγ agonists by a PPARγ pharmacophore–based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPARγ receptor binding. The neolignans bound to the PPARγ LBD with EC50 values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPARγ-mediated, but not human PPARα- or -β/δ-mediated luciferase reporter expression, with a pattern suggesting partial PPARγ agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPARγ expression. In conclusion, we identified neolignans as novel ligands for PPARγ, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.
doi:10.1124/mol.109.062141
PMCID: PMC3523390  PMID: 20064974
19.  Agonist-promoted Lys 63-linked polyubiquitination of the human kappa opioid receptor is involved in receptor down-regulation 
Molecular pharmacology  2008;73(4):1319-1330.
Ubiquitination of the human κ opioid receptor (hKOR) expressed in CHO cells was observed in the presence of the proteasomal inhibitor MG132 and enhanced by the agonists U50,488H and dynorphin A (Dyn A). The dominant negative (DN) mutants GRK2-K220R and β-arrestin (319–418), but not dynamin I-K44A, reduced Dyn A–stimulated hKOR ubiquitination and a phosphorylation-defective hKOR mutant (hKOR-S358N) did not undergo Dyn A-stimulated ubiquitination, indicating that hKOR ubiquitination is enhanced by receptor phosphorylation, but not by receptor internalization. A hKOR mutant (hKOR-10 KR) in which all ten intracellular Lys residues were changed to Arg showed greatly reduced basal and agonist-promoted receptor ubiquitination and substantially decreased Dyn A–induced receptor down-regulation, without changing ligand binding affinity, receptor-G protein coupling or receptor internalization or desensitization. The ubiquitination sites were further determined to be the three Lys residues in the C-terminal domain. The K63R ubiquitin mutant decreased Dyn A-induced hKOR ubiquitination and down-regulation, but the K48R mutant did not. Expression of HN-CYLD, a DN mutant of the deubiquitinating enzyme CYLD that breaks Lys 63-linked polyubiquitin chain, increased Dyn A-induced hKOR ubiquitination and down-regulation. These results indicate that ubiquitinated hKOR following agonist treatment contains predominantly Lys63-linked polyubiquitin chains and ubiquitination of the hKOR involved in agonist-induced down-regulation.
doi:10.1124/mol.107.042846
PMCID: PMC3489932  PMID: 18212250
20.  125I-4-(2-[7-Amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol, a High Affinity Antagonist Radioligand Selective for the A2a Adenosine Receptor 
Molecular pharmacology  1995;48(6):970-974.
SUMMARY
The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}-{1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5′-N-carboxamidoadenosine, 5′-N-ethylcarboxamidoadenosine, and (−)-N6-[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM241385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM241385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.
PMCID: PMC3479638  PMID: 8848012
21.  A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor 
Molecular pharmacology  1994;45(6):1101-1111.
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 rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.
PMCID: PMC3479652  PMID: 8022403
22.  A New High Affinity, Iodinated Adenosine Receptor Antagonist as a Radioligand/Photoaffinity Crosslinking Probe 
Molecular pharmacology  1987;32(1):184-188.
SUMMARY
A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenyl-acetylamino)ethyl]amino]carbonyl]-methyl]oxy]phenyl]-1,3-di-propylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD= 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125IPAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA > S-PIA > NECA ≫ IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.
PMCID: PMC3478951  PMID: 3614192
23.  The A2 Adenosine Receptor: Guanine Nucleotide Modulation of Agonist Binding Is Enhanced by Proteolysis 
Molecular pharmacology  1991;39(2):130-135.
SUMMARY
Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125l-2-[4-(2-{2-[(4-ammnophenyl)methylcarbonylamino]ethylaminnocarbonyl}ethyl)phenyl]ethylamino-5′-N-ethylcarboxamidoadenosine (1251-PAPA-APEC) and its photoaffinity analog 125l-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is Iabeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125l-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr ~47,O00 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory Gs-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit stnatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5′-(β,γ-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein.
Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the Gs protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.
PMCID: PMC3463105  PMID: 1899902
24.  A Functionalized Congener Approach to Adenosine Receptor Antagonists: Amino Acid Conjugates of 1,3-Dipropylxanthine 
Molecular pharmacology  1986;29(2):126-133.
SUMMARY
1,3-Dipropyl-8-phenylxanthine, a synthetic analog of theophylline and a potent antagonist of adenosine at A1 and A2-adenosine receptors, has been attached covalently through a functionalized chain to amino acids and oligopeptides. The xanthine conjugates have been studied as competitive inhibitors of the specific binding of [3H]N6-cyclohexyladenosine to A1-receptors of rat cerebral cortical membranes and for inhibition of cyclic AMP accumulation elicited by 2-chloroadenosine in guinea pig brain slices through A2-receptors. A free amino group on the extended chain generally resulted in high potency at A1-receptors. The potency (in some cases extending into the subnanomolar range) and selectivity for A1-receptors (up to 200-fold) suggest that this approach can yield a versatile class of “functionalized congeners” of adenosine receptor antagonists in which distal modifications of the attached moiety (“carrier”) can serve also to improve pharmacodynamic and pharmacokinetic parameters. The water solubility in many of the more potent analogs has been enhanced by two orders of magnitude over that of simple, uncharged 8-phenyl xanthine derivatives. Analogs in which the carrier contains d-tyrosine have potential for development of iodinated radioligands for adenosine receptors. The functionalized congener approach is potentially applicable to other drugs and for development of prodrugs.
PMCID: PMC3459325  PMID: 3005825
25.  Glycoprotein Nature of the A2-Adenosine Receptor Binding Subunit 
Molecular pharmacology  1990;38(2):177-183.
SUMMARY
Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by covalent labeling with the photoaffinity cross-liking ligand 125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonyl-amino]ethylaminocarbonyl]ethyl]phenyl]ethylamino-5′-N-ethyl-carboxamidoadenosine or directly with the azide derivative described in this paper. The protein comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding subunit from bovine brain were compared by partial peptide maps and following treatment with exo- and endoglycosidases. Peptide maps using two separate proteases reveal that the A1- and A2-adenosine receptor binding subunits share no common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000 without intermediate peptides, suggesting a single N-linked carbohydrate chain. The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an increased mobility on gel electrophoresis, suggesting the receptors contain a glycan component containing terminal sialic acid. Treatment of the labeled A2AR with α-mannosidase reveals two distinct populations of A2ARs, one of which is sensitive and the other resistant to the enzyme. The nonadditivity of sequential treatments with the two exoglycosidases suggests, a heterogeneous population of A2AR containing either complex- or high mannose-type carbohydrate chains. These data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate chain of either the complex or high mannose type. In addition, the A1- and A2ARs are distinct glycoproteins, as evidenced by their differing molecular weights (before and after deglycosylation) and distinct peptide maps.
PMCID: PMC3454504  PMID: 2385230

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