Interaction of the D-2short dopamine receptor with G proteins: analysis of receptor G protein selectivity

The human D-2short (D-2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [H-3]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [S-35]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [S-35]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D-2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([H-3]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [H-3]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [H-3]NPA binding with greater potency (similar to6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D-2S/Go preparation. [H-3]Spiperone binding occurred with an increased B-max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D-2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. (C) 2003 Elsevier Science Inc. All rights reserved.

Inverse agonism of the antipsychotic drugs at the D-2 dopamine receptor

The antipsychotic drugs had been assumed to act as antagonists at D-2 dopamine receptors but recently these drugs have been shown to possess inverse agonist properties at this receptor. Inverse agonism may be demonstrated from the ability of these drugs to potentiate forskolin-stimulated cAMP accumulation or to suppress agonist-independent [S-35]GTPgammaS binding. The antipsychotic drugs tested generally appear as full inverse agonists in these assays regardless of chemical or therapeutic class. The mechanism of inverse agonism of the antipsychotic drugs is still unclear but may involve stabilisation of the ground state of the D-2 receptor. (C) 2003 Elsevier Science B.V All rights reserved.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

Binding studies of a protonated dioxatetraazamacrocycle with carboxylate substrates

The tetraprotonated form of the dioxatetraazamacrocycle, 6,19-dioxa-3,9,16,22-tetraaza[22.2.2.2(11,14)]-triaconta-1(26),11,13,24, 27,29-hexaene, (H4L1)(4+), was used as the receptor for binding studies with carboxylate anionic substrates of different shapes, sizes, and charges [succinate (suc(2-)), cyclo- hexanetricarboxylate (cta(3-)), phthalate (ph(2-)), isophthalate (iph(2-)), terephthalate (tph(2-)), and benezenetricarboxylate (btc(3-))]. Association constants were determined by potentiometry in aqueous solution at 298.2 K and 0.10 M KCl and by H-1 NMR titration in D2O. The strongest association was found for the btc3- anion at 5-7 pH region. From both techniques it was possible to establish the binding preference trend of the receptor for the different substrates, and the H-1 NMR spectroscopy gave important suggestions about the type of interactions between partners and the location of the substrates in the supramolecular entities formed. The effective binding constants at pH 6 follow the order: btc(3-)>iph(2-)>cta(3-) =ph(2-)>tph(2-)>suc(2-). All the studies suggest that the anionic substrates bind to the receptor via N-H center dot center dot center dot O = C hydrogen bonds and electrostatic interactions, and the aromatic substrates can also establish pi-pi stacking interactions. The crystal structures of (H4L1)(4+) and its supramolecular assemblies with ph(2-) and tph(2-) were determined by X-ray diffraction. The last two structures showed that the association process in solid state occurs via multiple N-H center dot center dot center dot O = C hydrogen bonds with the anionic substrate located outside the macrocyclic cavity of the receptor. Molecular dynamics simulations carried out for the association of (H4L1)(4+) with tph(2-) and btC(3-) in water solution established at atomic level the existence of all interactions suggested by the experimental studies, which act cooperatively in the binding process. Furthermore, the binding free energies were estimated and the values are in agreement with the experimental ones, indicating that the binding of these two anionic substrates occurs into the receptor cavity. However, the tph(2-) has also propensity to leave the macrocyclic cavity and its molecular recognition can also happen at the top of the receptor. (C) 2008 Elsevier Ltd. All rights reserved.

Supramolecular aggregates between carboxylate anions and an octaaza macrocyclic receptor

The 28-membered octaazamacrocycle Me-2[28]py(2)N(6) was used as a receptor for the molecular recognition of aromatic and aliphatic carboxylate substrates. The receptor-substrate binding behaviour of (H6Me2[28]py(2)N(6))(6+) with an aliphatic (-O2C(CH2)(n)CO2-, n=0 to 4) and an aromatic (phthalate, isophthalate, terephthalate, 4,4'-dibenzoate, benzoate, 3- and 4-nitrobenzoate) series of carboxylate anions was evaluated by H-1 NMR spectroscopy (carried out in DMSO-d(6) at 300 K). Two association constants were found for most of the studied cases, except for 3- and 4-nitrobenzoate for which only K-1 was determined. For oxalate, malonate, benzoate and dibenzoate anions only the beta(2) constants could be obtained. The values of the first association constant cover a range from 2.86 to 3.69 (log units), and the second stepwise constant from 2.15 to 2.89 (also in log units). No special selectivity was found but the highest values were determined for adipate and the lowest for the monoprotic 3- and 4-nitrobenzoates. Single crystal X-ray structures of H6Me2[28]py(2)N(6)(6+) with terephthalate, 1, and 4,4'-dibenzoate (2) were determined showing supramolecular entities with general formula (H6Me2[28]py(2)N(6)).(substrate)(2)(PF6)(2).4H(2)O. These anions are the building blocks of an extensive 3-D network of hydrogen bonds.

Synthesis and evaluation of a solid supported molecular tweezer type receptor for cholesterol

A new macroporous stationary phase bearing 'tweezer' receptors that exhibit specificity for cholesterol has been constructed from rigid multifunctional vinylic monomers derived from 3,5-dibromobenzoic acid, propargyl alcohol and cholesterol. The synthesis of the novel tweezer monomer that contains two cholesterol receptor arms using palladium mediated Sonogashira methodologies and carbonate couplings is reported. The subsequent co-polymerisation of this tweezer monomer with a range of cross-linking agents via a 'pseudo' molecular imprinting approach afforded a diverse set of macroporous materials. The selectivity and efficacy of these materials for cholesterol binding was assessed using a chromatographic screening process. The optimum macroporous stationary phase material composition was subsequently used to construct monolithic solid phase extraction columns for use in the selective extraction of cholesterol from multi-component mixtures of structurally related steroids.

High-affinity small molecule-phospholipid complex formation: Binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction Of X-PA = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Oligomers of D-2 dopamine receptors: evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands

Mechanisms of G protein activation via the D-2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation

Background and purpose: The aim of this report is to study mechanisms of G protein activation by agonists. Experimental approach: The association and dissociation of guanosine 5'-O-(3-[S-35] thio) triphosphate ([S-35] GTP gamma S) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D-2short receptor was studied in the presence of a range of agonists. Key results: Binding of [S-35] GTPgS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D-2 receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [S-35] GTPgS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [S-35] GTPgS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [S-35] GTPgS binding was reduced with longer association times and increased [S-35] GTPgS concentrations. Conclusions and implications: These data are consistent with [S-35] GTPgS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [S-35] GTPgS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

Assays for enhanced activity of low efficacy partial agonists at the D-2 dopamine receptor

Background and purpose: Low efficacy partial agonists at the D-2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [S-35]GTP gamma S binding. Experimental approach: Agonist efficacy was assessed from [S-35]GTP gamma S binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[H-3] spiperone competition binding assays. Key results: When [S-35]GTP gamma S binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP-but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. Conclusions and implications: Substitution of Na+ by NMDG increases sensitivity in [S-35] GTPgS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Antagonists of the chemokine receptor, CCRS, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCRS (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [S-3]GTP gamma S binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2) -stimulated [S-35]GTP gamma S binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (similar to 15%) in cell surface CCRS was detected. (C) 2007 Elsevier Inc. All rights reserved

Probing the receptor interactions of an H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed. (C) 2007 Elsevier Ltd. All rights reserved.