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Mammalian sera contain binding proteins that specifically complex with somatomedins (insulin-like growth factor I and II) so that there are no detectable free somatomedins. There is immunologic evidence of two distinct types of serum binding proteins. The major binding protein complex (150,000 Mr) is growth hormone dependent. Binding proteins protect somatomedins from proteolytic degradation, retard plasma clearance, and decrease the availability to tissue receptors. The presence of serum binding proteins interferes with radioimmunoassays and radioreceptor assays for somatomedins. Proposed strategies to neutralize the interference from binding proteins without their elimination do not achieve this goal. Extraction of somatomedins by acid ethanol, hydrophobic absorption on C18 silicates (SepPak), and acid gel filtration are effective with human serum, but only acid gel filtration is satisfactory with rat serum. Failure to eliminate binding proteins from assays can lead to serious artifacts in conditions where abnormalities of binding proteins exist.
To obtain drugs which are more selective at benzodiazepine (BZD) receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine (compound A) was chosen for design and synthesis of new triazole derivatives as GABAA BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [3H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [3H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 μg cortical membrane protein and 8.6 × 10-5 nmole 3H-flumazenil in a final volume of 0.5 mL Tris-HCl buffer (50 mM, pH 7.4) at 30 °C. The binding parameters of [3H]-flumazenil, Bmax and Kd were determined through saturation studies as 0.638 ± 0.099 pmol/mg and 1.35 ± 0.316 nM respectively. The affinity of compound A was 1.9 nM comparable with diazepam (1.53nM). This finding makes the compound an interesting lead for further optimization. Starting from this compound, new ligands were synthesized and screened in-vitro by competitive binding assays.
Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.
This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff of a general hospital providing treatment to patients with severe COVID-19 were assayed for SARS-CoV-2 nucleocapsid protein (N) IgG using RLA. Nine patients with COVID-19 who had been treated in inpatient settings and had already recovered were used as control subjects, and 186 blood donor samples obtained more than 10 years ago were used as negative controls. Four of the 1000 samples showed apparently positive results, and approximately 10 or more samples showed slightly high counts. Interestingly, a few among the blood donor samples also showed slightly high values. To validate the results, antibody examinations using ELISA and neutralizing antibody tests were performed on 21 samples, and chemiluminescence immunoassay (CLIA) was performed on 201 samples, both resulting in a very high correlation. One blood donor sample showed slightly positive results in both RLA and CLIA, suggesting a cross-reaction. This study showed that five months after the pandemic began in Japan, the staff of a general hospital with a tertiary emergency medical facility had an extremely low seroprevalence of the antibodies against SARS-CoV-2. Further investigation will be needed to determine whether the slightly high results were due to cross-reactions or a low titer of anti-SARS-CoV-2 antibodies. The quantitative RLA was considered sensitive enough to detect low titers of antibodies.
Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D2-like receptors belonging to the D3 and D4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D3 receptor agonist [3H]7-hydroxy-N, N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D3 receptor. However, the selectivity of this compound for the D3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [3H]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D2-like receptor subtypes. [3H]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant (Kd) value of 0.32 + 0.03 nM and a maximum density of binding sites (Bmax) of 18.2 + 0.8 fmol/2 x 10(6) cells. [3H]7-OH-DPAT binding was unaffected by antibodies against dopamine D2 and D2S receptors. Anti-dopamine D3 and D4 receptor antibodies reduced [3H]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D3 and D4 receptor antibodies reduced remarkably [3H]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [3H]7-OH-DPAT labels dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.
[(3)H]4-DAMP is a radioligand that has been used to quantify levels of the muscarinic receptor CHRM3 protein in situ. However, in addition to high affinity binding to CHRM3, [(3)H]4-DAMP binds with low affinity to CHRM1 confounding the potential to discriminate between changes in these two muscarinic receptors. We have developed a [(3)H]4-DAMP binding assay, optimised for measuring CHRM3 protein levels in the cortex, with minimal selectivity towards CHRM1. The selectivity of our assay towards CHRM3 was confirmed using recombinant receptor-expressing, cell lysate preparations. [(3)H]4-DAMP binding levels were similar between wildtype and CHRM1 knockout mice, confirming that the amount of [(3)H]4-DAMP binding to CHRM1 was negligible. We used this assay to measure CHRM3 protein levels in the frontal pole, obtained post-mortem from subjects with bipolar disorder (n = 15), major depressive disorder (n = 15) and matched controls (n = 20) and showed that [(3)H]4-DAMP binding was not altered in either bipolar disorder or major depressive disorder. Western blotting confirmed that CHRM3 protein levels were unchanged in these subjects.
Adenosine receptors are involved in many physiological processes and pathological conditions and are therefore attractive therapeutic targets. To identify new types of effective ligands for these receptors, a library of adenosine derivatives bearing a boron cluster or phenyl group in the same position was designed. The ligands were screened in silico to determine their calculated affinities for the A2A and A3 adenosine receptors. An virtual screening protocol based on the PatchDock web server was developed. In the first screening phase, the effects of the functional group (organic or inorganic modulator) on the adenosine ligand affinity for the receptors were determined. Then, the lead compounds were identified for each receptor in the second virtual screening phase. Two pairs of the most promising ligands, compounds 3 and 4, and two ligands with lower affinity scores (compounds 11 and 12, one with a boron cluster and one with a phenyl group) were synthesized and tested in a radioligand replacement assay for affinity to the A2A and A3 receptors. A reasonable correlation of in silico and biological assay results was observed. In addition, the effects of a phenyl group and boron cluster, which is new adenosine modifiers, on the adenosine ligand binding were compared.
Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBAs) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [(3)H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the K(d) value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other K(d) value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5.
1. We have characterized the binding of the new potent and selective antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o[1,5- c]pyrimidine, [3H]-SCH 58261, to human cloned A2A adenosine receptors. 2. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]-SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (KD = 2.3 nM) and limited capacity (apparent Bmax = 526 fmol mg-1 protein). 3. Competition experiments revealed that binding of 0.5 nM [3H]-SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) = 2-phenylaminoadenosine (CV 1808) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > R-N6-phenylisopropyladenosine (R-PIA) > or = N6-cyclohexyladenosine (CHA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine receptor antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > SCH 58261 > xanthine amine congener (XAC) > (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3- dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > theophylline. 5. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. 6. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]-CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.
Sphingosine-1-phosphate (S1P) is a bioactive lipid that affects a variety of cellular processes through both its actions as a second messenger and via activation of a family of G protein-coupled receptors (S1P(1-5)). The study of S1P receptor pharmacology, particularly S1P(4), has been hindered by the lack of high-affinity radioligands with good specific activity. The studies presented herein characterize [(3)H]DH-S1P as a stable, high-affinity radioligand for S1P(4) pharmacology. Using a transfected Ba/F3 cell line selected for high hS1P(4) surface expression, we compared the consequences of different cellular backgrounds and commercial sources of sphingophospholipids on S1P(4) characterization. The development and subsequent use of the assay described has enabled us to extensively and definitively characterize the pharmacology of the human S1P(4) receptor.
1. We have recently suggested the existence in the heart of a 'putative beta4-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta1- and beta2-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[3H]-CGP 12177A ([-]-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[3H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand. 2. Increasing concentrations of (-)-[3H]-CGP 12177A, in the absence or presence of 20 microM (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta1- and beta2-adrenoceptors (pKD 9.4+/-0.1, Bmax 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta4-adrenoceptor' (pKD 7.5+/-0.1, Bmax 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta1- and beta2-adrenoceptors, (-)-[3H]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pKD 7.6+/-0.1, Bmax 50.8+/-7.4 fmol mg(-1) protein). 3. The non-conventional partial agonists (-)-CGP 12177A (pKi 7.3+/-0.2), (+/-)-cyanopindolol (pKi 7.6+/-0.2), [-]-pindolol (pK1 6.6+/-0.1) and (+/-)-carazolol (pKi 7.2+/-0.2) and the antagonist (-)-bupranolol (pKi 6.6+/-0.2), all competed for (-)-[3H]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta4-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4. The catecholamines competed with (-)-[3H]-CGP 12177A at the 'putative beta 4-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pKiH 6.3+/-0.3, pKiL 3.5+/-0.1), (-)-adrenaline (pKiH 6.5+/-0.2, pKiL 2.9+/-0.1), (-)-isoprenaline (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1), (+)-isoprenaline (pKi< 1.7), (-)-RO363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy++ +)-2-propranol)oxalate, pKi 5.5+/-0.1). 5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta4-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[3H]-CGP 12177A for one receptor state in the absence (pKi 7.3+/-0.2) or presence of GTP (pKi 7.3+/-0.2). (-)-Isoprenaline competed with (-)-[3H]-CGP 12177A for two states in the absence (pKiH 6.6+/-0.3, pKiL 3.5+/-0.1; % H 25+/-7) or presence of GTP (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1; % H 37+/-6). In contrast, at beta1-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6. The specificity of binding to the 'putative beta 4-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta 3-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]phenoxy]acetic acid, 6 microM), SR 58611A (ethyl{(7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl2-yloxy} acetate hydrochloride, 6 microM), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 microM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 microM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 microM) caused less than 22% inhibition of (-)[3H]-CGP 12177A binding in the presence of 500 nM (-)propranolol. Histamine (1mM), atropine (1 microM), phentolamine (10 microM), 5-HT (100 microM) and the 5-HT4 receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10nM) caused less than 26% inhibition of binding. 7.Non-conventional partial agonists, the antagonist (-)bupranolol and catecholamines all competed for (-)[3H]-CGP 12177A binding in the absence of (-)propranolol at beta1-adrenoceptors, with affinities (pKi) ranging from 1.6-3.6 log orders greater than at the 'putative beta 4-adrenoceptor'. 8.We have established and validated a radioligand binding assay in rat atrium for the 'putative beta 4-adrenoceptor' which is distinct from beta1-, beta2- and beta 3-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta 4-adrenoceptor'.
The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described (11)C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, (3)H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. (3)H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand (125)I-α-bungarotoxin ((125)I-BTX) in autoradiography, and unlabeled NS14492 displaced (125)I-BTX with an inhibition constant (Ki) of 23nM. (3)H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/mg protein. This binding assay further revealed the Ki rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of (3)H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that (3)H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.
Ciguatera, a global issue, lacks adequate capacity for ciguatoxin analysis in most affected countries. The Caribbean region, known for its endemic ciguatera and being home to a majority of the global small island developing states, particularly needs established methods for ciguatoxin detection in seafood and the environment. The radioligand receptor binding assay (r-RBA) is among the in vitro bioassays currently used for ciguatoxin analysis; however, similarly to the other chemical-based or bioassays that have been developed, it faces challenges due to limited standards and interlaboratory comparisons. This work presents a single laboratory validation of an r-RBA developed in a Cuban laboratory while characterizing the performance of the liquid scintillation counter instrument as a key external parameter. The results obtained show the assay is precise, accurate and robust, confirming its potential as a routine screening method for the detection and quantification of ciguatoxins. The new method will aid in identifying high-risk ciguatoxic fish in Cuba and the Caribbean region, supporting monitoring and scientific management of ciguatera and the development of early warning systems to enhance food safety and food security, and promote fair trade fisheries.
Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form. Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing α2A- and α2C-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the koff constants for various unlabeled ligands. The results show that the α2C-selectivity of MK912 depends mainly on a very slow off-rate at the α2C-adrenoceptor subtype. Regarding the markedly α2C- over α2A-selective compound spiroxatrine, its much faster on-rate at α2C- than α2A-adrenoceptors explains much of its exceptional α2C-selectivity. Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining koff constants of unlabeled drugs.
Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A3R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the Ki of eight different A3R antagonists, using CHO-K1 cells stably expressing the hA3R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off, as well as the dedicated K d of the A3R agonist [125I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds.
Both enantiomers of [(18)F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer's disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (-)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[(18)F]flubatine (specific activity > 350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[(18)F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.
A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor (CB1) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified CB1 were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid Δ9-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to CB1 were determined to be (from highest to lowest) 9.52 × 10-13 M (JWH-210), 6.54 × 10-12 M (JWH-250), 1.56 × 10-11 M (Δ9-tetrahydrocannabinol), 2.75 × 10-11 M (RCS-4), and 6.80 ×10-11 M (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.
Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.
The present study describes a modified radioreceptor binding assay using brain homogenate or serum from drug treated animals as the 'competing drug' in a conventional in vitro radioligand binding assay. Method validation involved measurement of the brain and serum concentration of three adenosine receptor antagonists following systemic administration, using a [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) binding assay. The intrinsic [3H]DPCPX binding capacity of test samples was abolished by protein denaturation (80 degrees C, 15 min) and, endogenous ligand was depleted enzymatically, prior to determination of drug concentration. Brain and serum concentrations of the adenosine A1 receptor antagonist, DPCPX increased in a dose related manner when measured 20 min after intraperitoneal injection. Estimated brain concentrations were 13.8, 87.7 and 288 nM following injection of 0.01, 0.1 and 1.0 mg/kg DPCPX, and serum concentrations were 26.5, 195 and 1370 nM respectively. A time dependent decrease in both brain and serum concentration was noted 20-180 min following injection of 1.0 mg/kg DPCPX. The peripheral adenosine receptor antagonists, 1,3-dipropyl-8-p-sulphophenylxanthine (DPSPX; 5.6 mg/kg) and 8-(p-sulphophenyl)theophylline (8-PST; 20 mg/kg), were not detected in brain tissue 20 min after intraperitoneal injection, despite serum concentrations of 56 and 52 microM respectively. This assay provides a useful and versatile method for determining the central penetration of neuroactive drugs.
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