Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791-2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain delta-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist ( approximately 10 nm); the lowest when receptor was bound to an antagonist ( approximately 500 nm); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of approximately 60 nm. Furthermore, GTP binding to the receptor.G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the Galphao (Kd approximately 20 nm) and lowest affinity toward the Galphai2 ( approximately 590 nm) subtypes, whereas the agonist-bound state had highest affinity for the Galphao and Galphai2 subtypes (Kd approximately 9 nm and approximately 7 nm, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.     

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).     

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.     

Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.     

Binding of agonists and antagonists to muscarinic receptors

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [³H] acetylcholine. Mechanisms that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

Highly potent and selective zwitterionic agonists of the delta-opioid receptor. Part 1

A series of zwitterionic delta-opioid agonists, with targeted physicochemistry, as a strategy to limit potential for CNS exposure, were prepared. These agents were found to possess exquisite potency and selectivity over mu and kappa-opiate activity. Furthermore, analogue 3a was found to display restricted CNS exposure, as evidenced by its inactivity in a rodent hyperlocomotion assay of central opiate activity. Dog pharmacokinetic studies on 3a indicated encouraging oral bioavailability.     

Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB₁ and CB₂ receptors and were identified as new lead structures. In further GTPγS binding studies selected compounds were shown to be antagonists or inverse agonists.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Sulfhydryl groups are involved in the binding of agonists and antagonists to the human mineralocorticoid receptor

To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4°C inhibited the binding to hMR of both [³H]aldosterone and [³H]RU26752 (a synthetic aldosterone antagonist). At 4°C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4°C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20°C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hMR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action.     

Identification of agonists and antagonists of the human melanocortin-4 receptor from piperazinebenzylamines

SAR studies of a series of piperazinebenzylamines resulted in identification of potent agonists and antagonists of the human melanocortin-4 receptor. Thus, the 1,2,3,4-tetrahydroisoquinolin-1-ylacetyl compound 12e and the quinolin-3-ylcarbonyl analogue 12l possessed K(i) values of 6.3 and 4.5 nM, respectively. Interestingly, 12e was a full agonist with an EC(50) value of 31 nM, and 12l was a weak partial agonist (IA=17%) and functioned as an antagonist (IC(50)=300 nM).     

Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization.

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.     

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.     

Differential sorting of human delta-opioid receptors after internalization by peptide and alkaloid agonists

Desensitization and internalization of G protein-coupled receptors observed after agonist activation are considered two important regulatory processes of receptor transduction. Endogenous human delta-opioid receptors (hDOR) are differentially regulated in terms of desensitization by peptide ([d-Pen2,5]enkephalin (DPDPE) and Deltorphin I) and alkaloid (etorphine) agonists in the neuroblastoma cell line SK-N-BE (Allouche, S., Roussel, M., Marie, N., and Jauzac, P. (1999) Eur. J. Pharmacol. 371, 235-240). In the present study, we examined the role of hDOR internalization and down-regulation in this differential desensitization. Sustained activation by peptides for 30 min caused a marked decrease of both [3H]diprenorphine binding sites and hDOR immunoreactivity, observed in a Western blot, whereas a moderate reduction by 30% was observed after a 30- and 60-min etorphine exposure in binding experiments without opioid receptor degradation. Using fluorescence microscopy, we visualized hDOR internalization promoted by different agonists in SK-N-BE cells expressing FLAG-tagged hDOR. Agonist withdrawal results in a greater recycling process correlated with a stronger hDOR resensitization after etorphine treatment compared with DPDPE or Deltorphin I, as shown in binding, immunocytochemical, and functional experiments. This suggests a distinct sorting of opioid receptors after their internalization. We demonstrated a lysosomal hDOR targeting upon peptides by using chloroquine in binding, Western blot, and immunocytochemical experiments and by colocalization of this receptor with a late endosome marker. In contrast, when the recycling endosome blocker monensin was used, acceleration of desensitization associated with a strong intracellular immunostaining was observed upon etorphine treatment. The possibility of separate endocytic pathways responsible for the differential sorting of hDOR upon peptide and alkaloid ligand exposure was ruled out by binding and immunocytochemical experiments using sucrose hypertonic solution. First, these results showed complex relationships between hDOR internalization/down-regulation and desensitization. Second, we demonstrated for the first time that the same receptor could undergo a distinct sorting after internalization by peptide and alkaloid agonists.     

Conformational states of the nicotinic acetylcholine receptor from Torpedo californica induced by the binding of agonists, antagonists, and local anesthetics. Equilibrium measurements using tritium-hydrogen exchange

The tritium-hydrogen exchange kinetics of Torpedo californica AChR, in native membrane vesicles at pH 7.4 and 0 degrees C, have been analyzed in the presence of agonists, partial agonists, local anesthetics, and competitive antagonists. The agonists carbamylcholine (10 microM-1 mM) and suberyldicholine (10 microM) and the partial agonists decamethonium (25 microM and 1 mM) and hexamethonium (1 mM) have no effect on the exchange kinetics, although at lower concentration carbamylcholine may slightly accelerate exchange. Nondesensitizing local anesthetics do affect the exchange behavior, dependent on concentration. Procaine at 500 microM moderately retards exchange while procaine at 10 mM and tetracaine at 5 mM slightly accelerate exchange. The competitive antagonist alpha-bungarotoxin retards exchange significantly, as does d-tubocurarine although to a lesser extent. These results suggest that the resting and desensitized conformations of the AChR are very similar in overall solvent accessibility and that at lower concentrations noncompetitive blockers such as procaine may stabilize a less solvent-accessible state of the AChR. The competitive antagonists alpha-bungarotoxin and d-tubocurare also stabilize a dynamically restricted, less solvent-accessible conformation of the acetylcholine receptor, demonstrating that a large conformational change accompanies binding of these toxins. Any change in conformation which may accompany desensitization is very different from these effects.     

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.     

Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states

The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.     

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists. Part 2. Versatile steroidal carboxamide derivatives

To further proceed with our previous work, novel steroid-based histamine H₃ receptor antagonists were identified and characterized. Using an ‘amine-to-amide’ modification strategy at position 17, in vitro and in vivo potent monoamino steroid derivatives were found during the lead optimization. Usage of the non-basic amide moiety resulted in beneficial effects both in activity and selectivity. The 15α-carboxamido derivative 10 was not only highly active at human and rat H₃ receptors, but also showed negligible activity at rat muscarinic receptors. Furthermore, it proved to be considerably stable in human and rat microsomes and showed significant in vivo potency in the pharmacodynamic rat dipsogenia test and in the water-labyrinth cognitive model. Based on all of these considerations, compound 10 was appointed to be a preclinical candidate.     

Phe-D-Leu-Phe-D-Leu-Phe derivatives as formylpeptide receptor antagonists in human neutrophils: cellular and conformational aspects.

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.