Potent Inhibitor Of Binding Research Articles

Intrapulmonary IFN-γ instillation causes chronic lymphocytic inflammation in the spleen and lung through the CXCR3 pathway

Some patients with chronic refractory cough have high levels of pulmonary IFN-γ and IFN-γ-producing T lymphocytes. Pulmonary IFN-γ administration causes acute airway lymphocytic inflammation and cough hypersensitivity by increasing the number of pulmonary IFN-γ-producing T lymphocytes, but these lymphocytes may be recruited from other organs. Intraperitoneal IFN-γ injection can increase the spleen weight of mice. It remains elusive whether pulmonary IFN-γ can induce chronic airway lymphocytic inflammation and cough hypersensitivity by stimulating the proliferation of IFN-γ -producing T lymphocytes in the spleen. Here, we found that pulmonary IFN-γ administration induced chronic airway inflammation and chronic cough hypersensitivity with an increased number of IFN-γ-producing T lymphocytes in the spleen, blood and lung. Pulmonary IFN-γ administration also increased 1) the proliferation of spleen lymphocytes in vivo and 2) the IP-10 level and CXCR3+ T lymphocyte numbers in the spleen and lung of mice. IP-10 could promote the proliferation of spleen lymphocytes in vitro but not blood lymphocytes or lung-resident lymphocytes. AMG487, a potent inhibitor of binding between IP-10 and CXCR3, could block pulmonary IFN-γ instillation-induced chronic airway lymphocytic inflammation and the proliferation of IFN-γ-producing T lymphocytes in mouse spleens. In conclusion, intrapulmonary IFN-γ instillation may induce the proliferation of splenic IFN-γ-producing T lymphocytes through IP-10 and the CXCR3 pathway. The IFN-γ-producing T lymphocytes in blood, partly released from the mouse spleen, may be partly attracted to the lung by pulmonary IP-10 through the CXCR3 pathway. IFN-γ-producing T lymphocytes and IFN-γ in the lung may cause chronic airway lymphocytic inflammation and chronic cough hypersensitivity.

Synthesis and Evaluation of a 18F-Labeled Triazinediamine Analogue for Imaging Mutant IDH1 Expression in Gliomas by PET.

Mutations in the isocitrate dehydrogenase gene 1 (IDH1) are common in gliomas. Studies suggest that IDH1 mutations are early events in glioma formation and are important drivers of malignant progression. Herein, we report the synthesis and evaluation of a 18F-labeled triazinediamine analogue, [18F]1, as a candidate radiotracer for noninvasive imaging of IDH1 mutations in gliomas by positron emission tomography (PET). In vitro studies revealed good binding inhibition potency and binding affinity for [18F]1 in IDH1 mutant glioma cell lines, with a half-maximal inhibitory concentration value (IC50) of 54 nM and an equilibrium dissociation constant (Kd) of 40 nM. In vivo studies using mutant IDH1 glioma xenografts showed good tumor uptake of [18F]1 and specific inhibition by the unlabeled 1, but also elevated radioactivity uptake in the bone, suggesting significant defluorination. The results support further optimization of the triazinediamine scaffold to develop a more stable and potent 18F-labeled analogue for PET imaging of IDH1 mutations in gliomas.

Inhibition of Delta-induced Notch signaling using fucose analogs.

Notch is a cell-surface receptor that controls cell fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools for inhibiting Notch activity are being developed as cancer therapeutics. Towards this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

Pharmacologic Profile of Naloxegol, a Peripherally Acting µ-Opioid Receptor Antagonist, for the Treatment of Opioid-Induced Constipation

Opioid-induced constipation (OIC) is a common side effect of opioid pharmacotherapy for the management of pain because opioid agonists bind to µ-opioid receptors in the enteric nervous system (ENS). Naloxegol, a polyethylene glycol derivative of naloxol, which is a derivative of naloxone and a peripherally acting µ-opioid receptor antagonist, targets the physiologic mechanisms that cause OIC. Pharmacologic measures of opioid activity and pharmacokinetic measures of central nervous system (CNS) penetration were employed to characterize the mechanism of action of naloxegol. At the human µ-opioid receptor in vitro, naloxegol was a potent inhibitor of binding (Ki = 7.42 nM) and a neutral competitive antagonist (pA2 - 7.95); agonist effects were <10% up to 30 μM and identical to those of naloxone. The oral doses achieving 50% of the maximal effect in the rat for antagonism of morphine-induced inhibition of gastrointestinal transit and morphine-induced antinociception in the hot plate assay were 23.1 and 55.4 mg/kg for naloxegol and 0.69 and 1.14 mg/kg by for naloxone, respectively. In the human colon adenocarcinoma cell transport assay, naloxegol was a substrate for the P-glycoprotein transporter, with low apparent permeability in the apical to basolateral direction, and penetrated the CNS 15-fold slower than naloxone in a rat brain perfusion model. Naloxegol-derived radioactivity was poorly distributed throughout the rat CNS and was eliminated from most tissues within 24 hours. These findings corroborate phase 3 clinical studies demonstrating that naloxegol relieves OIC-associated symptoms in patients with chronic noncancer pain by antagonizing the µ-opioid receptor in the ENS while preserving CNS-mediated analgesia.

The CMG2 ELISA for evaluating inhibitors of the binding of anthrax toxin protective antigen to its receptor

Introduction Anthrax toxin comprises a protective antigen (PA) of MW 83 kDa, a lethal factor (LF) and an edema factor (EF). Upon binding to its receptor on cell surfaces, PA 83 is enzymatically cleaved to a 63 kDa product (PA 63), followed by binding of LF or EF, receptor-mediated internalisation of these factors, and production of their toxic effects. The high-affinity binding of PA 83 to its receptor is essential for the intoxication process. To study the interaction between the PA and its receptor, and inhibition of the binding, an enzyme-linked immunosorbent assay (ELISA) was developed. Methods One of the two known anthrax toxin receptors (capillary morphogenesis factor 2; CMG2) was adsorbed onto wells of a 96-well plate. Either PA 83 or PA 63 was then added to the receptor-coated wells, followed by sequential addition of anti-PA antibody, anti-species antibody-enzyme conjugate, and enzyme substrate at appropriate time intervals. Results Best results were obtained by overnight incubation of CMG2 in PBS at 4 °C. CMG2 was used at 1 μg/ml because of the cost of the commercial product. The rate of change of absorbance was low, and was measured over 3 h to obtain accurate results. The assay results increased almost linearly with CMG2 concentration to 10 μg/ml. PA 83 was also used at 1 μg/ml, but the assay values reached a plateau at approx. 10 μg/ml. Binding was divalent cation-dependent, almost irreversible, and free CMG2 was a potent inhibitor of binding ( I 50 in the nM range). Binding of PA 63 was similar to that of PA 83. Discussion The high-affinity binding and divalent cation dependence confirm the validity of the assay as a model for toxin-receptor binding in vivo and as a means of evaluating toxin-receptor binding and inhibitors of the binding. Attempts to use crude lysates of J774A.1 cells or von Willebrand factor as an alternative source of anthrax toxin receptor were not successful.

Effect of amino acid modifications on the molecular structure of adsorbed and nonadsorbed bombesin 6–14 fragments on an electrochemically roughened silver surface

AbstractThis work used infrared absorption and Raman spectroscopy to determine the structure of seven modified fragments (residues 6–14 of the polypeptide chain) of bombesin (BN6–14). The peptides studied are cyclo[D‐Phe6, His7, Leu14]BN6–14, [D‐Phe6, Leu‐NHEt13, des‐Met14]BN6–14, [D‐Phe6, Leu13‐®‐p‐chloro‐Phe14]BN6–14, [D‐Phe6, β‐Ala11, Phe13, Nle14]BN6–14, [D‐Tyr6, β‐Ala11, Phe13, Nle14]BN6–14, [D‐Tyr6, β‐Phe11, Phe13, Nle14OH]BN6–14 and [D‐Cys6, Asn7, D‐Ala11, Cys14]BN6–14. These peptides are potent bombesin agonists useful in the treatment of tumors.Surface‐enhanced Raman scattering (SERS) spectroscopy was also used to examine the behavior of these molecules on an electrochemically roughened silver surface. The SERS spectra reveal that substituting native amino acids in these molecules with synthetic ones changes their adsorption state slightly on an electrochemically roughened surface of silver. The peptides [D‐Tyr6, β‐Ala11, Phe13, Nle14]BN6–14 and [D‐Tyr6, β‐Phe11, Phe13, Nle14OH]BN6–14 tend to adsorb strongly on this surface via C fragment (∼1400 cm−1). The observed medium enhancement of the Trp8 residue and amide bond Raman signals indicate further interactions between these fragments and the surface. [D‐Phe6, Leu‐NHEt13, des‐Met14]BN6–14 and [D‐Cys6, Asn7, D‐Ala11, Cys14]BN6–14 are shown to be coordinated to the silver through CONH, CO, and the indole ring. The strongest SERS bands (∼1506, ∼1275, ∼1149, and ∼1007 cm−1) of [D‐Phe6, Leu13‐®‐p‐chloro‐Phe14]BN6–14 and [D‐Phe6, β‐Ala11, Phe13, Nle14]BN6–14 suggest that these two peptides bind to the silver via Trp8 and CONH. In the case of cyclo[D‐Phe6, His7, Leu14]BN6–14, the formation of a peptide/Ag complex is confirmed by the strong SERS bands involving Trp8 and CONH vibrations, which are accompanied by a SERS signal due to the CO vibrations.For these analogs, the relative potency for inhibition of binding of 125I‐[Tyr4]BN to rat pancreas acini cells was correlated with the behavior of the amide bond on the silver surface, while the contribution of the structural components to the ability to interact with the rGRP‐R was correlated with the SERS patterns. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept.

Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their H(CC) (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.

Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury

Acute pancreatitis is a common, and as yet incurable, clinical condition, the incidence of which has been increasing over recent years. Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that treatment with a neutralizing antibody against CINC, a CXC chemokine, protects rats against acute pancreatitis-associated lung injury. The hexapeptide antileukinate (Ac-RRWWCR-NH 2) is a potent inhibitor of binding of CXC chemokines to the receptors (CXCR2). This study aims to evaluate the effect of treatment with antileukinate on acute pancreatitis and the associated lung injury in mice. Acute pancreatitis was induced in adult male Swiss mice by hourly intra-peritoneal injections of caerulein (50 μg/kg/h) for 10 h. Antileukinate (52.63 mg/kg, s.c.) was administered to mice either 30 min before or 1 h after starting caerulein injections. Severity of acute pancreatitis was determined by measuring plasma amylase, pancreatic water content, pancreatic myeloperoxidase (MPO) activity, pancreatic macrophage inflammatory protein-2 (MIP-2) levels and histological examination of sections of pancreas. A rise in lung MPO activity and histological evidence of lung injury in lung sections was used as criteria for pancreatitis-associated lung injury. Treatment with antileukinate protected mice against acute pancreatitis and associated lung injury, showing thereby that anti-chemokine therapy may be of value in this condition.

Radioligand binding and functional responses of ligands for human recombinant adenosine A3 receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.

Dissociation of high-affinity cocaine analog binding and dopamine uptake inhibition at the dopamine transporter.

Cocaine initiates its euphoric effects by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. It has been hypothesized that the DAT transmembrane aspartic acid residue D79 forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine. We examined the consequences of novel and previously studied mutations of the D79 residue on DAT uptake of [3H]dopamine, DAT binding of the cocaine analog [3H]WIN 35,428, and drug inhibition of each process, all under identical conditions. The rat D79E DAT mutation decreased dopamine uptake Vmax by 7-fold and decreased dopamine turnover by 4-fold. Wild-type DAT displayed near-perfect agreement in the uptake and binding inhibition potencies for substrates, but cocaine and other nonsubstrate inhibitor drugs were approximately 3-fold less potent in uptake than in binding assays. Apparent affinities for substrates were unaffected by the D79E mutation unless the catechol moiety was modified. Strikingly, potencies for nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT, because of a 3-fold lowering of binding affinities relative to WT DAT. The present findings reveal a complex role for D79 in determining substrate specificity and high-affinity binding of DAT inhibitors. We propose that at least two discrete inhibitor-binding DAT conformations or populations exist and that the DAT conformation/population responsible for inhibitor high-affinity binding is less responsible for dopamine uptake. The findings may be extensible to other psychostimulants and antidepressants that display discrepancies between binding affinity and monoamine uptake inhibition potency and may be relevant to development of a long-sought "cocaine antagonist".

Thrombospondin-1 binds to polyhistidine with high affinity and specificity

Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.

Thrombospondin-1 binds to polyhistidine with high affinity and specificity

Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a 125I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of 125I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a Ki of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.

Cyclopentapeptides as Flexible Conformational Templates

Studies of 3D models for cyclopentapeptides (CPP's) employing only NMR spectroscopy encounter a serious problem. Because of conformer averaging, 3D structure(s) derived directly from NMR data may not correspond to the energy minimum (minima) with low relative conformational energy. At the same time, independent energy calculations can determine all low-energy conformers for the CPP backbone. The two approaches are compared in this study by results obtained for cyclo(d-Pro1-Ala2-Ala3-Ala4-Ala5). Contrary to the conclusion (predominance of the βII‘γ type conformer) of earlier NMR studies, independent energy calculations found a different family of low-energy 3D structures that are consistent both with the NMR data in DMSO and with the known X-ray data on CPP's. The preferable Ala4 conformations were found in the αR/αL regions suggesting studies of cyclo(d-Pro1-Ala2-Ala3-Aib4-Ala5) which was synthesized. Further NMR studies confirmed the results of the independent energy calculations. The independent energy calculations have been applied also to cyclo(Arg1-Gly2-Asp3-d-Phe4-Val5) and cyclo(Arg1-Gly2-Asp3-Phe4-d-Val5). Both peptides are almost equally potent inhibitors of binding of αIIbβ3 integrins to fibrinogen and of αVβ3 integrins to vitronectin. If both of them possess a NMR-predicted conformer of the βII‘γ type, however, the conformations of the active sequence, Arg1-Gly2-Asp3, should be dissimilar in these two peptides. This discrepancy is eliminated in the 3D pharmacophore model proposed by independent energy calculations. The model is also in good agreement with the model by other authors that was confirmed by X-ray studies.

Affinity of cholecystokinin receptor antagonists for the gastrin-binding protein

A 78 kDa gastrin-binding protein is a likely target for the anti-proliferative effects of the cholecystokinin (CCK) receptor antagonists d, l-4-benzamido- N, N-dipropylglutaramic acid (proglumide) and N-4-chlorobenzoyl- l-tryptophan (benzotript) on colorectal carcinoma cell lines [Baldwin, G.S., 1994. Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein. Proc. Natl. Acad. Sci. USA 91, 7593–7597.]. Definition of the physiological role of the gastrin-binding protein has been hampered by the very low affinity of benzotript for the gastrin-binding protein. Benzotript analogues were therefore tested for their ability to inhibit the binding of iodinated gastrin to the gastrin-binding protein. The affinity of the most potent analogue (the d-isomer of benzotript, CR 665) was similar to the value reported previously for the l-isomer. In order to isolate more potent binding inhibitors, several selective CCK receptor antagonists were also tested as inhibitors of the binding of gastrin to the gastrin-binding protein. The affinity of the most potent binding inhibitor PD 149164 (benzenebutanoic acid, 4-fluoro-!b/-[[3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo-[3.3.1.1 3,7]dec-2-yloxy)carbonyl]amino]propyl]amino]-, [ R-( R*, S*)]-) was approximately 10-fold higher than the l-isomer of benzotript. PD 149164 may serve as the lead compound for the future development of more potent and selective gastrin-binding protein inhibitors.

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin’s binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy–entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.

Actions of butyrophenones and other antipsychotic agents at NMDA receptors: relationship with clinical effects and structural considerations

Haloperidol inhibits NMDA receptors with higher affinity for NMDA receptors composed of NR1/2B compared with NR1/2A. To assess whether the clinical effects of haloperidol and other antipsychotic agents are mediated through this site on NMDA receptors and to examine structure activity relationships at this site, we examined the ability of a variety of drugs with neuroleptic actions to inhibit NMDA receptor function. Many antipsychotic agents inhibit 125I-MK 801 binding to the NMDA receptor with IC 50values in the micromolar range. The rank order of potency for inhibition of binding to adult rat forebrain was trifluperidol (TFP) > clozapine = fluphenazine = reduced haloperidol = spiperone = trifluoperazine = butaclamol ≫ pimozide = risperidone = sulpiride. These findings match the molecular biological specification of the agents, with trifluperidol having a marked preference for NR1/2B (ε2) receptors. Mutations at ε2E201, which alter the effects of haloperidol, also decrease the affinity of TFP but not other modulators is mediated by this residue of the NMDA receptor. The present results demonstrate that while TFP acts on NMDA receptors in a mannor simmilar to haloperidol, other antipsychotic agents do not share the specific pharmacological properties of this action, suggesting that their clinical mechanism is not mediated by this receptor.

RETRACTED: GlcNAc-terminated glycodendrimers form defined precipitates with the soluble dimeric receptor of rat natural killer cells, sNKR-P1A

Synthetic GlcNAc-terminated thiourea-bridged glycoclusters were found to be potent inhibitors of binding of the soluble dimeric receptor of rat natural killer cells, sNKR-P1A protein, to its high affinity ligand. Moreover, we have shown here that characteristic precipitation curves can be recorded upon mixing of the GlcNAc glycoclusters with sNKR-P1A. For the GlcNAc8 glycocluster the precipitation curve is biphasic, with high affinity and low affinity precipitates differing in their sensitivity towards GlcNAc-mediated inhibition of precipitation. Quantitative analyses of the precipitates indicate the occurrence of a single sugar binding site per sNKR-P1A subunit, and lead to a model of the most possible spatial arrangements of the glycocluster-receptor lattices. These results provide new tools for further studies on carbohydrate recognition by NKR-P1A.

Erinacine E as a kappa opioid receptor agonist and its new analogs from a basidiomycete, Hericium ramosum.

A kappa opioid receptor binding inhibitor was isolated from the fermentation broth of a basidiomycete, Hericium ramosum CL24240 and identified as erinacine E (1). Three analogs of 1 were produced by fermentation in other media and by microbial biotransformation. Of these compounds, 1 was shown to be the most potent binding inhibitor. Preliminary SAR studies of these compounds indicated that all functional groups and side chains were required for the activity. Compound 1 was a highly-selective binding inhibitor for the kappa opioid receptor: 0.8 microM (IC50) for kappa, >200 microM for mu, and >200 microM for delta opioid receptor. Compound 1 suppressed electrically-stimulated twitch responses of rabbit vas deferens with an ED50 of 14 microM. The suppression was recovered by adding a selective kappa opioid receptor antagonist nor-binaltorphimine, indicating that 1 is a kappa opioid receptor agonist.

Nucleotide regulation and characteristics of potassium channel opener binding to skeletal muscle membranes.

[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.

A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines.

Melanoma growth stimulatory activity (MGSA/GROalpha) is a 73 amino acid peptide sharing sequence characteristics with the alpha-chemokine superfamily. MGSA/GROalpha is produced by diverse melanoma cell lines and reported to act as an autocrine growth factor for the cells. We tested the binding of MGSA/GROalpha to melanoma cell lines, Hs 294T and RPMI7951, and found that these cells could bind to MGSA/GROalpha but not to interleukin-8. Recently, we defined a novel hexapeptide, antileukinate, which is a potent inhibitor of binding of alpha-chemokines to their receptors on neutrophils. When antileukinate was added to melanoma cells, it inhibited the binding of MGSA/ GROalpha. The growth of cells from both melanoma cell lines was suppressed completely in the presence of 100 microM peptide. The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of the excess amount of MGSA/GROalpha. The viability of Hs 294T cells in the presence of 100 microM peptide was > 92%. These findings suggest that MGSA/GROalpha is an essential autostimulatory growth factor for melanoma cells and antileukinate inhibits their growth by preventing MGSA/GROalpha from binding to its receptors.