Analysis of receptor-G protein interactions in permeabilized cells

Receptor-induced binding of the stable GTP analogue, guanosine 5-[-thio]triphosphate (GTP [S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus -toxin, binding of GTP [S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.Overall, this study indicates that receptor-stimulated GTP [S] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.     

Appropriate calibration curve fitting in ligand binding assays

Calibration curves for ligand binding assays are generally characterized by a nonlinear relationship between the mean response and the analyte concentration. Typically, the response exhibits a sigmoidal relationship with concentration. The currently accepted reference model for these calibration curves is the 4-parameter logistic (4-PL) model, which optimizes accuracy and precision over the maximum usable calibration range. Incorporation of weighting into the model requires additional effort but generally results in improved calibration curve performance. For calibration curves with some asymmetry, introduction of a fifth parameter (5-PL) may further improve the goodness of fit of the experimental data to the algorithm. Alternative models should be used with caution and with knowledge of the accuracy and precision performance of the model across the entire calibration range, but particularly at upper and lower analyte concentration areas, where the 4- and 5-PL algorithms generally outperform alternative models. Several assay design parameters, such as placement of calibrator concentrations across the selected range and assay layout on multiwell plates, should be considered, to enable optimal application of the 4- or 5-PL model. The fit of the experimental data to the model should be evaluated by assessment of agreement of nominal and model-predicted data for calibrators.     

Differential regulation of 5-HT1A receptor-G protein interactions in brain following chronic antidepressant administration.

Changes in 5-HT(1A) receptor function or sensitivity following chronic antidepressant treatment may involve changes in receptor-G protein interaction. We have examined the effect of chronic administration of the SSRI fluoxetine or the tricyclic antidepressant amitriptyline on 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in serotonergic cell body areas, and cortical and limbic structures using quantitative autoradiography. Treatment of rats with fluoxetine, but not amitriptyline, resulted in an attenuation of 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the dorsal and median raphe nuclei. The binding of the antagonist radioligand [3H]MPPF to 5-HT(1A) receptor sites was not altered, suggesting that the observed changes in 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding were not due to changes in receptor number. Thus, the desensitization of somatodendritic 5-HT(1A) autoreceptors in the dorsal and median raphe following chronic SSRI treatment appears to be due to a reduced capacity of the 5-HT(1A) receptor to activate G protein. By contrast, no significant change in postsynaptic 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding was observed in any of the forebrain areas examined following chronic antidepressant treatment. Thus, changes in postsynaptic 5-HT(1A) receptor-mediated responses reported to follow chronic SSRI or tricyclic antidepressant administration most likely occur distal to receptor-G protein interaction, perhaps at the level of effector, or involving changes in neuronal function at the system or circuit level.     

Non-selectivity of amitriptyline for subtypes of brain muscarinic receptors demonstrated in binding and functional assays

The characteristics of interaction of amitriptyline, a tricyclic antidepressant, with rat brain muscarinic receptors were assessed using both radioligand binding and functional assays. In competition studies, amitriptyline displaced muscarinic ligand binding from a single high-affinity site in homogenates of various brain regions which have a different distribution of M1 and M2 receptor subtypes. The affinity of amitriptyline for muscarinic receptors was also comparable in all brain regions. Furthermore, amitriptyline identified a single species of muscarinic receptors in intact cells dissociated from the cerebral cortex and in cerebrocortical slices. The non-selectivity of amitriptyline for muscarinic receptor subtypes in these preparations was in contrast to the selectivity exhibited by pirenzepine. This non-selective nature of amitriptyline was also evident in functional assays, since this antidepressant was equipotent at antagonizing M1-mediated increase in phosphoinositide hydrolysis and M2-mediated inhibition of cyclic AMP formation in dissociated cortical cells. Atropine was also equipotent at blocking both responses but was 20- to 30-fold more potent than amitriptyline. These results demonstrate that amitriptyline behaves as a non-selective muscarinic antagonist using both radioligand binding and functional measurements.     

Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

Background and Purpose

Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889.

Experimental Approach

UCB1244283 was characterized in vitro using radioligand binding assays with [³H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice.

Key Results

Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [³H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [³H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions.

Conclusions and Implications

These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies.     

Temperature effect on serum protein binding kinetics of phenytoin in monotherapy patients with epilepsy.

The effects of temperature on the binding kinetics of phenytoin (PHT) to serum proteins were determined in patients with epilepsy. Serum samples examined in the study were obtained from 59 patients (31 male, 28 female) with epilepsy on PHT monotherapy. Their age ranged from 3 to 64 years (mean (SD), 23.3 (16.3) years). Protein binding of PHT was evaluated by ultrafiltration under current routine laboratory conditions (25 +/- 3 degrees C) or at a temperature of 37 degrees C. The in vivo binding parameters of PHT to serum proteins were determined using a binding equation derived from the Scatchard equation for a one-site binding model. Significant differences were observed in serum concentrations of unbound PHT between paired data (P < 0.05). The mean association constant (K) of PHT to serum proteins is 0.011 microM-1 at 25 +/- 3 degrees C and 0.006 microM-1 at 37 degrees C, while mean total concentration of binding sites (n(Pt)) is 1002 microM for 25 +/- 3 degrees C and 1112 microM for 37 degrees C. Significant differences were observed in the binding kinetics of PHT to serum proteins for the different temperature conditions of ultrafiltration (P < 0.05). Our study confirms that binding affinity for PHT-serum protein interaction is approximately 45% lower at 37 degrees C than at 25 +/- 3 degrees C and consequently, binding potential (K.n(Pt)) is approximately 39% lower at 37 degrees C than at 25 +/- 3 degrees C.     

Effects of short-term toluene exposure on ligand binding to muscarinic acetylcholine receptors in the rat frontal cortex and hippocampus

Changes in the binding affinity of the muscarinic acetylcholine receptor agonist carbamylcholine were determined in membranes isolated from the brains of rats exposed to toluene at concentrations of 500-2,000 ppm for 6 h. Membrane fractions of the frontal cortex and hippocampus were prepared and agonist-binding affinities were determined by measuring the displacement of [3H]N-methyl scopolamine-binding activity by carbamylcholine. In the frontal cortex, the affinity of high-affinity carbamylcholine binding was reduced following exposure to toluene at a concentration of 1000 ppm or higher. However, in the hippocampus, the affinity of high-affinity binding of carbamylcholine was increased following exposure to toluene. These observations suggest that toluene exposure affects binding affinity of carbamylcholine, and the effect differs by brain region.     

High-throughput screening assays to discover small-molecule inhibitors of protein interactions

The availability of large collections of small-molecule inhibitors of protein interactions would bear a tremendous impact both on academic and therapeutic research. The past recent years have seen a marked acceleration in the discovery of protein interaction inhibitors, through structure-based drug design but mostly through screening efforts. This article attempts to review the impressive number and variety of in vitro and cellular screening assays that have been developed and, for most of them, used successfully to identify small-molecule inhibitors of protein interactions. Various strategies aimed at improving hit rates are also reviewed, and future challenges to improve discovery success rates are discussed. The growing list of protein interaction inhibitors and the large arsenal of screening methods, now available to most laboratories or screening facilities, will probably convince an increasing number of academic and industrial scientists that protein interactions are more druggable than once feared, and that their respective research interests would greatly benefit from the discovery of protein interaction inhibitors.     

Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract Guanine nucleotide effects on agonist binding

Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 microM) and KD-low ( approximately 10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 microM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.     

The effect of oxime reactivators on muscarinic receptors: functional and binding examinations

The antidotal treatment of organophosphorus poisoning is still a problematic issue since no versatile antidote has been developed yet. In our study, we focused on an interesting property, which does not relate to the reactivation of inhibited acetylcholinesterase (AChE) of some oximes, but refers to their anti-muscarinic effects which may contribute considerably to their treatment efficacy. One standard reactivator (HI-6) and two new compounds (K027 and K203) have been investigated for their antimuscarinic properties. Anti-muscarinic effects were studies by means of an in vitro stimulated atrium preparation (functional test), the [(3)H]-QNB binding assay and G-protein coupled receptor assay (GPCR, beta-Arrestin Assay). Based on the functional data HI-6 demonstrates the highest anti-muscarinic effect. However, only when comparing [(3)H]-QNB binding results and GPCR data, K203 shows a very promising compound with regard to anti-muscarinic potency. The therapeutic impact of these findings has been discussed.     

Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification

Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions.     

Changes in muscarinic ligand binding to intestinal muscle strips produced by pre-exposure to hypotonic conditions.

The extent of the binding of [3H]propylbenzilylcholine mustard (3H-PrBCM) to muscarinic receptors in longitudinal muscle strips from guinea-pig small intestine is increased by nearly 50% when the strips are preexposed to distilled water before measurement of 3H-PrBCM binding in Krebs-Henseleit solution. The apparent rate constant for 3H-PrBCM-receptor complex formation is more than double that of intact strips. The curves for the inhibition of 3H-PrBCM binding by methylatropinium bromide in normal and treated strips are superimposable, but, in contrast, distilled water pretreatment shifts the inhibition curve for carbachol to lower concentrations by a factor of 5-6. The inhibition curve for methylfurmethide is also shifted, by a factor of approximately 4, but the effect on the curve for hexyltrimethylammonium (C6TMA) is slight. The relative inhibition produced by benzhexol in the two preparations was variable. Comparison of the rate of equilibration of benzhexol with muscarinic receptors in intact and in distilled water pretreated muscle indicates that this inconsistency is unlikely to be due to incomplete equilibration.     

A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor

THRX-160209 is a potent antagonist at the M(2) muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M2 receptor subtype (apparent pK(I) = 9.51 +/- 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK(I) values < 6.0), 2) specificity of THRX-160209 for the M2 receptor subtype compared with the closely related M4 (apparent pK(I) = 8.78 +/- 0.24) and M1, M3, and M5 receptors (apparent pK(I) values 10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [3H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.     

Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding

Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical and nonsymmetrical compounds was synthesized in which the phthalimide residues were replaced by differently substituted imide moieties. The allosteric action was measured in porcine heart muscarinic M(2) receptors using [(3)H]N-methylscopolamine (NMS) as a ligand for the orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range, while the inhibition of NMS binding was maintained. Additional propyl chain methylation was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain in allosteric activity achieved by ring variation plus propyl chain methylation on one side of the molecule could not be augmented by symmetrical variations. The elevation of the ligand binding can be explained by different quantitative structure-activity relationships for the affinities to the free and the orthoster-liganded receptor.     

The effect of sulphydryl block on the binding of H1-antagonists to the muscarinic receptor.

The treatment of brain muscarinic receptors by p-chlormercuribenzoate is known to change structure-activity relationships for antagonists. The changes in binding affinity of H1-antagonists for this receptor have been measured; the changes are similar to those for intrinsic muscarinic antagonists.     

In-gel protein phosphatase assays and other useful methods for the detection of protein phosphatase activities

Intracellular signaling is governed by protein phosphorylation and dephosphorylation catalyzed by protein kinases and protein phosphatases, respectively. Since there is growing evidence that a variety of protein phosphatases are involved in the pathogenesis of various diseases, protein phosphatases have recently been the focus of intense research interest, not only in basic biology but also in clinical medicine. In the process of these studies, analytical methods for protein phosphatases will be of increasing importance. A major bottleneck in protein phosphatase assays is the selection and preparation of an efficient substrate for the phosphatase to be assayed. To circumvent this difficulty, a variety of protein phosphatase substrates have been devised during the development of novel assay techniques by which protein phosphatase activities can be readily detected. In this review, we focus on the methodology for detecting protein phosphatase activities, with special emphasis on in-gel protein phosphatase assays and related techniques. The utility and limitations of these methods are also discussed.     

Transition and heavy metal inhibition of ligand binding to muscarinic acetylcholine receptors from rat brain.

Several divalent heavy metals interact with muscarinic acetylcholine receptors in neural membranes from rat forebrains in vitro to inhibit the the binding of [³H]3-quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. Mercury is the most potent inhibitor with an ID50 value of 10^?7m for the inhibition of the binding of 10^?10m QNB. Several other metals are less effective with ID50 values between 10^?5 and 2 × 10^?4m increasing in the following order: iron < silver < copper < lead < cadmium < terbium < zinc. Cobalt, manganese, lanthanum, nickel, and tin are less potent inhibitors (ID50 > 10^?3m). Binding inhibition by copper, mercury, and cadmium is competitive, and the inhibition by all the metals can be reversed by decreasing the free metal concentration through dilution or chelation by ethylenediaminetetraacetate, dimercaprol, or penicillamine. The effects of metals on muscarinic binding are largely independent of temperature. Chaotropic anions (100 mm iodide, thiocyanate, trichloroacetate, or nitrate) are similarly without effect on the metal-receptor interaction. The limited effects of sulfhydryl reducing (dithiothreitol, 2-mercaptoethanol, and glutathione) and alkylating (N-ethyl maleimide) reagents do not indicate a prominent role for sulfhydryl groups in mediating metal inhibition of muscarinic receptors.     

Clinical significance of drug binding,protein binding,and binding displacement drug interactions

Drugs widely used in clinical psychopharmacology, with few exceptions, are hepatically eliminated and circulate in the blood bound to plasma proteins. Often, the degree of binding is high (>90%). This fact has led to a widespread belief that such drugs may frequently participate in drug-binding interactions. The logic is as follows: Coadministration of drugs can result in a highly bound drug displacing a less avidly bound drug from its binding sites. This leads to greater amounts of free, nonprotein-bound, drug available for distribution to the sites of action. As free drug is a major determinant of pharmacologic effects, these drug interactions could result in toxicity and/or enhanced efficacy. This reasoning simplifies a complex situation where compensatory changes occur in the body to buffer the impact of drug-binding interactions. There are few examples where the above events have been documented to occur with psychoactive drugs leading to substantial clinical consequences. Although protein-binding displacement interactions are generally of minimal clinical significance, this is an assumption not based on evidence, but rather the lack of it. The purpose of this review is to examine some relevant aspects of drug-protein binding and draw conclusions for clinical practice about the significance of drug binding and drug-binding displacement interactions. Psychopharmacology Bulletin.     

Aging alters in a region-specific manner serotonin transporter sites and 5-HT(1A) receptor-G protein interactions in hamster brain

Key proteins regulating serotonergic activity, specifically the serotonin transporter and 5-HT(1A) receptor, were examined in the midbrain raphe nuclei of young (3-4 months) and old (17-19 months) hamsters (N=7-10/group). An age-related decrease in the maximal density of serotonin transporter sites labelled with [(3)H]paroxetine (fmol/mg protein, Old: 396+/-13; Young: 487+/-27) was observed in the dorsal raphe nucleus (DRN) but not the median raphe nucleus (MRN), without affecting the affinity of [(3)H]paroxetine. In the DRN and MRN, the stimulation of [(35)S]GTP gamma S binding by the 5-HT(1A) receptor agonist 8-OH-DPAT, or the number of 5-HT(1A) receptor sites labeled with [(3)H] MPPF, was not different in old versus young animals. Thus in the DRN, aging decreased serotonin transporter sites without changing 5-HT(1A) receptor activation of G proteins or 5-HT(1A) receptor density. In the CA(1) region of hippocampus, 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding was increased in the older animals (% above basal, Old: 141+/-21; Young: 81+/-17) without changing specific [(3)H] MPPF binding sites, suggesting that the capacity of 5-HT(1A) receptors to activate G proteins is enhanced. Aging also appears to enhance this capacity in the dentate gyrus, because this region exhibited a constant level of 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding in spite of an age-related decrease in the number of [(3)H] MPPF binding sites (fmol/mg protein, Old: 203+/-21; Young: 429+/-51).