Radiolabeled Ligand Binding Assays Research Articles

New characterization of dihydroergotamine receptor pharmacology in the context of migraine: utilization of a β-arrestin recruitment assay

IntroductionDihydroergotamine mesylate (DHE) is an established effective acute therapy for migraine and is often characterized by its broad receptor pharmacology. Knowledge of DHE pharmacology largely comes from studies employing older methodologies.ObjectiveTo assess DHE receptor activity using high-throughput methods to screen for functional ß-arrestin activity at G protein–coupled receptors (GPCRs).MethodsFunctional receptor activities of DHE and sumatriptan succinate (both 10 μM) were screened against 168 GPCRs using the gpcrMAX assay. Agonist and antagonist effects were considered significant if receptor activity was >30% or inhibited by >50%, respectively. Radiolabeled ligand binding assays were performed for DHE (0.01–300 nM for 5-HT3 and 4E; 0.3–10,000 nM for 5-HT1B, α-adrenergic2B [i.e., α2B-adrenoceptor], D2, and D5) to assess specific binding to select receptors.ResultsDHE (10 μM) exhibited agonist activity at α-adrenergic2B, CXC chemokine receptor 7 (CXCR7), dopamine (D)2/5, and 5-hydroxytryptamine (5-HT)1A/1B/2A/2C/5A receptors and antagonist activity at α-adrenergic1B/2A/2C (i.e., α1B/2A/2C-adrenoceptors), calcitonin receptor–receptor activity modifying protein 2 (CTR-RAMP2) or amylin 2 (AMY2), D1/3/4/5, and 5-HT1F receptors. Sumatriptan succinate (10 μM) exhibited agonist activity at the 5-HT1B/1E/1F/5A receptors. DHE demonstrated a half-maximal inhibitory concentration (IC50) of 149 nM at the 5-HT1F receptor and a half-maximal effective concentration (EC50) of 6 μM at the CXCR7 receptor. DHE did not bind to the 5-HT3 receptor at concentrations up to 300 nM and bound poorly to 5-HT4E and D5 receptors (IC50 of 230 and 370 nM, respectively). DHE bound strongly to the D2, 5-HT1B, and α-adrenergic2B receptors (IC50 of 0.47, 0.58, and 2.8 nM, respectively).ConclusionBy using a high-throughput β-arrestin recruitment assay, this study confirmed the broad receptor profile of DHE and provided an update on DHE receptor pharmacology as it relates to migraine.

1, 6-di-O-caffeoyl-β-D-glucopyranoside, a natural compound from Callicarpa nudiflora Hook impairs P2Y12 and thromboxane A2 receptor-mediated amplification of platelet activation and aggregation

BackgroundPlatelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-β-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation. PurposeThe aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms. MethodsThe experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αIIbβ3 integrin abundance, serotonin (5-HT) secretion, thromboxane A2 (TXA2) synthesis was determined to assess the effects of CGP on platelet activation. Furthermore, RhoA and PI3K/Akt/GSK3β signal transduction were analyzed by Western Blotting assay. In addition, radiolabelled ligand binding assay was involved to evaluate the ability of CGP binding to thromboxane prostanoid (TP) and P2Y12 receptors. ResultsCGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αIIbβ3 integrin activation, serotonin (5-HT) secretion from granule and thromboxane A2 (TXA2) synthesis. Next, we found that CGP suppressed RhoA and PI3K/Akt/GSK3β signal transduction. Data from radiolabelled ligand binding assay showed that CGP displayed apparent competing effects on TP and P2Y12 receptors. ConclusionCollectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y12 and TP receptors.

Paclitaxel Is an Inhibitor and Its Boron Dipyrromethene Derivative Is a Fluorescent Recognition Agent for Botulinum Neurotoxin Subtype A

We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol "protein scanning with virtual ligand screening" (PSVLS). Hit selection used an in-house developed holistic binding scoring method. Selected hits were tested experimentally for inhibitory activity using fluorescence resonance energy transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex through either radiolabeled ligand binding assays (nonfluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A's proteolytic activity experimentally with an IC50 of 5.2 μM. A fluorescent derivative was also confirmed to bind to the toxin and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multiprobe detection assays.

Potent in vitro and in vivo antitumor activity of interleukin‐4‐conjugated Pseudomonas exotoxin against human biliary tract carcinoma

Targeting cytotoxins or immunotoxins to tumor cell surface receptors represents a new approach for the treatment of cancers. We tested the antitumor activity of a cytotoxin (IL-4-PE) composed of an interleukin-4 (IL-4) targeting moiety and a truncated form of Pseudomonas exotoxin A against human biliary tract carcinoma (BTC). Immunohistochemical analysis showed that cultured BTC cell lines and cancerous epithelia in BTC tissue (e.g., gallbladder carcinoma, extrahepatic cholangiocarcinoma, and intrahepatic cholangiocarcinoma) expressed receptors for IL-4 in situ at high densities. However, normal epithelial cells in gallbladder and bile duct tissues did not express these IL-4 receptors. Eight BTC cell lines expressed IL-4R on the cell surface as determined by radiolabeled ligand binding assays. When these cells were treated with IL-4-PE, significant cytotoxicity was observed as determined by the inhibition of protein synthesis. The concentration of agent causing 50% inhibition of protein synthesis (IC(50)) was found to be less than 10 ng/mL in 4 of the 8 BTC cell lines studied. The antitumor activity of IL-4-PE was assessed for human BTC cells implanted subcutaneously in immunodeficient mice. By intratumoral injection of IL-4-PE, complete disappearance of the established tumors was observed in 40% of animals. Intraperitoneal administration of IL-4-PE at tolerated doses to animals with peritoneally disseminated BTC exhibited significantly prolonged survival compared to untreated animals (>14 weeks vs. 5 weeks in treated and untreated mice, respectively). These results indicate that IL-4 receptor-targeted cytotoxin is a potent agent that may provide a new therapeutic option for BTC.

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Détection des ciguatoxines : avantages et inconvénients des différentes méthodes biologiques utilisées

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17β-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17β-E-6-BSA to GAPDH was impeded by free estrogen (17β-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10× lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V max and changes in K m were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V max significantly. Testosterone, corticosterone, 17α-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17β-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.

The proliferative response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model

Objective: Previous studies on graft healing have shown increased platelet-derived growth factor (PDGF) production in graft segments versus native aortic segments. The purpose of this study was to characterize the proliferative response of graft smooth muscle cells (SMCs) to PDGF. Methods: Thoracoabdominal grafts were implanted in beagles. SMCs were harvested from the graft and the proximal and distal aortas. Basal proliferation was assessed with growth curves in primary culture. The proliferative response to PDGF then was compared with [ 3H]thymidine uptake studies and cell counts. Finally, PDGF receptors were characterized with radio-labeled ligand binding assays. Results: The growth curves showed that the graft SMCs entered log-phase growth 2 days earlier than did the aortic SMCs. Stimulation of quiescent early-passage graft SMCs with PDGF (10 ng/mL) resulted in a 1.7 ± 0.1–fold increase in [ 3H]thymidine incorporation, which was significantly less than that of the SMCs from both the proximal aorta (11.8 ± 3.0) and the distal aorta (10.2 ± 1.9; P < .5). Similarly, the 1.1 ± 0.1–fold increase in graft SMC cell number was significantly less than the increases for both proximal (2.8 ± 0.5) and distal (2.9 ± 0.8) aortic SMCs ( P < .5). Binding studies on quiescent first-passage cells showed fewer PDGF receptors available for binding in the graft SMCs (185 ± 70 fmol/million cells) as compared with both the proximal (419 ± 147 fmol/million cells) and the distal (387 ± 112 fmol/million cells) aortas ( P < .5). Binding affinity was similar for the three groups. Conclusion: Graft SMCs exist in a chronic proliferative state but exhibit a decreased proliferative response to PDGF and have fewer receptors available for binding PDGF than do aortic SMCs in vitro. (J Vasc Surg 1999;29:845-51.)

De novo design, synthesis, and biological activities of high-affinity and selective non-peptide agonists of the delta-opioid receptor.

On the basis of the structure-activity relationships of delta-opioid-selective peptide ligands and on a model of the proposed bioactive conformation for a potent and selective, conformationally constrained delta-opioid peptide ligand [(2S, 3R)-TMT1]DPDPE, a series of small organic peptide mimetic compounds targeted for the delta-opioid receptor have been designed, synthesized, and evaluated in radiolabeled ligand binding assays and in vitro bioassays. The new non-peptide ligands use piperazine as a template to present the most important pharmacophore groups, including phenol and phenyl groups and a hydrophobic moiety. This hydrophobic group was designed to mimic the hydrophobic character of the D-Pen residues in DPDPE, which has been found to be extremely important for increasing the binding affinity and selectivity of these non-peptide ligands for the delta-opioid receptor over the mu-opioid receptor. Compound 6f (SL-3111) showed 8 nM binding affinity and over 2000-fold selectivity for the delta-opioid receptor over the mu-opioid receptor. Both enantiomers of SL-3111 were separated, and the (-)-isomer was shown to be the compound with the highest affinity for the delta-opioid receptor found in our study (IC50 = 4.1 nM), with a selectivity very similar to that observed for the racemic compound. The phenol hydroxyl group of SL-3111 turned out to be essential to maintain high affinity for the delta-opioid receptor, which also was observed in the case of the delta-opioid-selective peptide ligand DPDPE. Binding studies of SL-3111 and [p-ClPhe4]DPDPE on the cloned wild-type and mutated human delta-opioid receptors suggested that the new non-peptide ligand has a binding profile similar to that of DPDPE but different from that of (+)-4-[((alphaR)-alpha(2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), another delta-opioid-selective non-peptide ligand.

Modulation of vitamin D receptor and estrogen receptor by 1,25(OH)2-vitamin D3 in T-47D human breast cancer cells

1,25(OH)2-Vitamin D3 inhibits breast cancer cell proliferation through interaction with the vitamin D receptor (VDR). Regulation of VDR is under the influence of several factors which include the functional ligand for this receptor (1,25(OH)2-vitamin D3) as well as heterologous steroid hormones. We evaluated the nature of homologous regulation in T-47D human breast cancer cells with a radiolabelled ligand binding assay and a ribonuclease protection assay for VDR. Significant VDR up-regulation, as measured by hormone binding assays, occurred with pre-incubations with 10(-9)M through 10(-6)M 1,25(OH)2-vitamin D3 (P < 0.05). A 7-fold VDR up-regulation with 10(-8)M 1,25(OH)2-vitamin D3 occurred at 4 h treatment and was not associated with an increase in VDR mRNA expression on ribonuclease protection assay. This supports the hypothesis that up-regulation of VDR is probably the result of ligand-induced stabilization of pre-existing receptor. All-trans-retinoic acid, the progesterone analog R-5020, and prednisone were found to induce heterologous up-regulation of the VDR. We then determined with ligand binding assays whether 1,25(OH)2-vitamin D3 could influence receptor levels for another hormone in a manner analogous to the heterologous regulation of VDR. Regulation of estrogen receptor (ER) by 1,25(OH)2-vitamin D3 was studied in T-47D and MDA-MB-231 breast cancer cells. Incubation of T-47D cells, which are ER (+), with 10(-8)M 1,25(OH)2-vitamin D3 did not result in up-regulation of ER. Yet estrogen binding was significantly up-regulated in a cell line that is ER(-), MDA-MB-231. The increased estrogen binding was associated with a shift in binding affinity and ribonuclease protection assay showed absence of ER mRNA in these cells, suggesting an up-regulation of estrogen binding proteins and not of the ER itself.

Interactions of cell-surface galactosyltransferase with immunoglobulins

Detection of the activity of β-1,4-galactosyltransferase (β-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of β-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of β-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface β-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of β-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-β-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn 2+ and UDP-[ 3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[ 3H]-galactose, β-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > α chain > μ chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, β-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the β-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, α-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37°C, the apparent affinity constants and association rate constants of interaction between cell surface β-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and α2 chain or monomeric IgA1 were in the range from 7.1 × 10 7 to 4.6 × 10 8 M −1 and from 1 × 10 5 to 3 × 10 6 M −1 s −1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 × 10 −2 to 5.0 × 10 −4 s −1 and from 33 to 1380 s, respectively. The number of α2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 × 10 5 to 1.3 × 10 6. The binding of IgA by the cell surface β-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

Pharmacologic Characterization of the Rabbit Neutrophil Receptor for Platelet-Activating Factor

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.

Interaction of Lipophorin with the Plasma Membrane of Locust Flight Muscles

Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.

Binding of lipophorin to the fat body of the migratory locust

The interaction of locust high density lipophorin (HDLp) with pieces of fat body tissue was studied at 33°C using a radiolabelled ligand binding assay. Under the assay conditions, binding of tritium-labelled HDLp ([ 3H]HDLp) was demonstrated to correlate linearly with tissue concentration up to ∼ 7 mg of fat body protein per ml of incubation medium. The [ 3H]HDLp binding that was displaceable by a 20-fold excess of unlabelled HDLp (which is an approximation of the specific binding) reached equilibrium after ∼ 2 h, whereas low levels of non-displaceable binding increased linearly during this time interval. Analysis of the concentration dependent total binding of [ 3H]HDLp revealed the presence of a specific binding site with an equilibrium dissociation constant of K d = 3.1 (±0.5) × 10 −7 M and a maximal binding capacity of 9.8 (±0.5) ng μg −1 tissue protein. Competition experiments demonstrated that the affinity of unlabelled HDLp for the binding site is similar to the affinity of [ 3H]HDLp. Unlabelled low density lipophorin (LDLp), however, was shown to have an approx. 20-fold lower affinity for the binding site.

Evidence for homogeneity of thromboxane A 2 receptor using structurally different antagonists

Nine structurally dissimilar thromboxane antagonists (SQ 29548, ICI 185282, AH 23848, BM 13505 (Daltroban), BM 13177 (Sulotroban), SK&F 88046, L-636499, L-640035 and a Bayer compound SK&F 47821) were studied for activity as thromboxane A 2 receptor antagonists. The assays used were inhibition of responses induced by the thromboxane mimetic, U46619, on human washed platelet aggregation, rabbit platelet aggregation, rabbit aortic strip contraction, anaesthetised guinea-pig bronchoconstriction, and a radio-labelled ligand ( 125I-PTA-OH) binding assay as a measure of affinity for the human platelet receptor. The results of the present study, with activities spanning at least four orders of magnitude along with statistically significant correlations (at least P < 0.01), strongly suggests that between assays, antagonists and species a homogenous population of thromboxane A 2 receptors exists. This finding is in contrast to those of a close series of 13-azapinane antagonists studied by other workers which have suggested receptor heterogeneity.