Non-Specific Binding of the Fluorescent B-Adrenergic Receptor Probe Alprenolol-NBD

Abstract

The fluorescent ß-adrenergic receptor probe alprenolol-NBD was found to exhibit a high affinity (K_d 3.2 nM) and a low capacity (10 fmol/mg protein) for the ß₂-adrenergic receptor on living Chang liver cells but also a high affinity (K_d 320 nM) for non-ß-adrenergic receptor binding sites with a very high capacity (28,000 fmol/mg protein). Calculations are presented which make clear that less than 3% of the binding of alprenolol-NBD during visualization experiments is ß-adrenergic receptor related. Furthermore, it is shown that besides the downregulation of ß-adrenergic receptors during incubation with isoproterenol, the high-affinity non-ß-receptor binding sites are also deminishing during incubation with isoproterenol. Based on our findings it is concluded that the results of Henis et al. (1) who claimed the visualization of the ß-adrenergic receptor population on Chang liver cells by alprenolol-NBD must be interpreted as an almost completely non-specific fluorescence.     

Design and synthesis of thiourea based receptor containing naphthalene as oxalate selective sensor

The thiourea based receptor containing naphthalene groups (1), has been successfully designed and synthesized for application as an oxalate receptor. A density functional theory at B3LYP/6-31G(d,p) level of theory has been applied to predict the binding ability between 1 and selected anions, i.e., oxalate, malonate, succinate, glutarate, dihydrogen phosphate, and hydrogen sulphate. Calculation results point out that receptor 1 shows the strongest interaction to oxalate ion with the binding free energy of 172.48 kcal mol⁻¹. The recognition ability of 1 to the selected anions has been also investigated by means of the absorption and emission techniques. Experimental results are in excellent agreement with the calculation data in which receptor 1 shows highly selective for oxalate ion over the other anions with logβ of 3.82 (0.02) M⁻¹ by means of the size of binding cavity.      

Design of Angiotensin II Derivatives Suitable for Indirect Affinity Techniques: Potential Applications to Receptor Studies

Abstract

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-All (Bio-Ahx-All) and dinitrophenyl- aminohexanoyl-All (Dnp-Ahx-All). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (K_d values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-All and Dnp-Ahx-All behaved as agonists on several All-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala¹,Phe(4N₃)⁸)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido All-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.     

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Background: Development of functional monoclonal antibodies against intractable GPCR targets.

Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.

Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.

Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.

Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large ‘family A’ of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor.

The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.     

Quantitative Receptor Autoradiography: Application to the Characterization of Multiple Receptor Subtypes

Abstract

In vitro autoradiographic techniques combined with computer assisted microdensitometry were used to analyze the characteristics and distribution of multiple recognition sites for the neurotransmitters acetylcholine (M₁ and M₂) and serotonin (5-HT_1A and 5-HT_1B). For this purpose, binding competition experiments were performed using non-subtype selective ³H-labeled ligands and selective unlabeled compounds. Consecutive tissue sections were incubated in the presence of increasing concentrations of displacers. By using this approach, maximal densities of binding sites, as well as competition profiles of several drugs could be analyzed and quantified in microscopic brain areas. Our results reveal the presence of brain structures enriched in one class of muscarinic or serotonergic-1 recognition sites. This provides a tool for better characterization of the proposed “subtype-selective” ligands and suggests physiological functions for these receptor subtypes. It is concluded that quantitative autoradiographic techniques provide a level of anatomical and pharmacological information on neurotransmitter receptor subtypes, which is difficult to attain using membrane binding studies.     

FitDockApp：基于模板的分子对接PyMOL插件图形化计算软件

目的 分子对接在预测分子之间的结合模式和亲和力方面起着至关重要的作用,是计算结构生物学和计算机辅助药物设计研究的重要方法。本研究团队近期开发了一款基于模板的新型对接方法FitDock,当存在近似的蛋白质配体模板时,它在准确性和速度方面都超过了业界常用的分子对接方法。为了增强FitDock方法的可用性,使其在分子模拟领域得到更广泛的应用,很有必要发展图像化的软件工具。方法 基于Python图像化编程,本文开发了FitDockApp,这是分子可视化软件PyMOL的插件软件。结果 FitDockApp能够通过操作窗口界面,实现基于模板的分子对接和配体结构比对,实时显示预测三维结构,并提供将对接文件上传到实验室服务器获取最优模板的便利。此外,FitDockApp还具备批量对接功能。结论 FitDockApp通过用户友好的界面简化了对接过程,并提供丰富的功能,帮助研究人员获得精确的对接结果。FitDockApp是一款免费软件,兼容Windows和Linux系统,可在http://cao.labshare.cn/fitdock/下载。     

Opiate Receptor Sites in the Rabbit Cerebellum: Autoradiographic Distribution

Abstract

The distribution of the [³H] diprenorphine binding sites in the rabbit cerebellum has been analyzed after specific labelling of tissue slices in vitro followed by autoradiohraphic processing. High, moderate and very low silver grain densities were observed over the molecular, granular and white layers, respectively. Within each layer, the distribution of the autoradiographic grain exhibited no evident patterning. Therefore, further work using complementary techniques is needed to determine the precise location of the opiate receptor sites in the rabbit cerebellum.     

Effect of Novel Amino Acids and Dipeptides Substituted 3-Morpholino Arecoline Derivatives as Muscarinic Receptor 1 Agonists in Alzheimer’s Dementia Models

A series of novel, potent and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester group of arecoline (which is reported as mucarinic agonist) has been replaced by N-substituted morpholine ring. The structure activity relationship reveals that the increase in lipophilic carbon chain on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor. In the present study, we are reporting N-amino acid substituted 9(a–k) and dipeptides substituted 10(a–j) and 11(a–j) morpholino arecoline derivatives, along with their in vitro muscarinic binding studies by using [³H]QNB and also in vivo evaluation of memory and learning in male Wistar rats (passive avoidance test plus maze studies) as M1 receptor agonist. Some molecules from the dipeptide series (10b, 10c and 10j) showed potent M1 receptor agonist activity. Other derivatives also showed considerable M1 receptor binding affinity.     

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid: rational design,synthesis and biological evaluation

A series of o-, m- and p-benzyl tetrazole derivatives 11a–c has been designed, synthesized and evaluated as potential Angiotensin II AT1 receptor antagonists, based on urocanic acid. Compound 11b with tetrazole moiety at the m-position showed moderate, however, higher activity compared to the o- and p-counterpart analogues. Molecular modelling techniques were performed in order to extract their putative bioactive conformations and explore their binding modes.     

Proximity probing assays for simultaneous visualization of protein complexes in situ

Evaluation of: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print).

Techniques for in situ detection and quantification of proteins in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer patients. Several techniques are currently available for detection of single proteins and post-translational modifications, but very few are suitable for detection of protein complexes. Methods that enable simultaneous detection and quantification of protein complexes provide novel possibilities for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research. Leuchowius et al. describe an improved proximity ligation assay for in situ detection of protein complexes, which is able to detect and quantify several protein complexes simultaneously in the same tissue specimen.     

Receptor Binding Profiles of Amiloride Analogues Provide no Evidence for a Link Between Receptors and the Na+/H+ Exchange,But Indicate a Common Structure on Receptor Proteins

Abstract

Amiloride and its analogues affect radioligand binding to the adenosine-A₁ receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na⁺ transport, in particular Na⁺/H⁺ exchange. The receptor binding profiles of amiloride, benzamil and 5′-(N,N-hexamethylene)amiloride (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na⁺/H⁺ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins.     

Investigation on the Potential Role of the AH Receptor Nuclear Translocator Protein in Vitamin D Receptor Action

Abstract

The Ah receptor nuclear translocator protein (ARNT) is required for binding of the Ah (dioxin) receptor to the xenobiotic responsive element (XRE), and is a structural component of the XRE-binding form of the Ah receptor. The vitamin D receptor requires an accessory protein for binding to the vitamin D responsive element (VDRE) in the osteocalcin gene. Since the vitamin D receptor has similarities to the Ah receptor, we investigated whether ARNT is also required for vitamin D receptor activity. Two lines of evidence demonstrate that ARNT is not required for vitamin D receptor activity, and therefore does not correspond to the vitamin D receptor accessory protein: i) Antibodies to ARNT have no effect on binding of the vitamin D receptor to the VDRE. ii) c4, a mutant of Hepa-1 cells that is defective in ARNT activity, and in which binding of the Ah receptor to the XRE does not occur, possesses a vitamin D receptor with full activity for binding the VDRE.     

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

BackgroundThe EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands.MethodsHere, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays.ResultsWe find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration.ConclusionsThe experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer.General significanceThis work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.     

5-HT Receptors: Subtypes and Second Messengers

Abstract

Our knowledge about 5-HT (serotonin, 5-hydroxytryptamine) receptors has gained significantly over the recent few years. The discovery of selective ligands and the use of new techniques have led to a significant increase in the number of recognised receptors subtypes. The present status of awareness is largely related to the use of radioligand binding studies, autoradiography, second messenger analysis and more recently, molecular biological techniques. Three main families of 5-HT receptors, of which subtypes have been described, are now accepted. This heterogeneity is further substantiated by the cloning of the cDNA's of three different 5-HT receptors. This article reviews some of the recent developments which led to the characterisation of 5-HT receptor subtypes.     

Identification of dual ligands targeting angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ by core hopping of telmisartan

It has been reported previously that some angiotensin II receptor blockers not only antagonize angiotensin II type 1 receptor (AT₁R), but also exert stimulation in peroxisome proliferator-activated receptor γ (PPARγ) partial activation, among which telmisartan displays the best. Telmisartan has been tested as a bifunctional ligand with antihypertensive and hypoglycemic activity. Aiming at more potent leads with selective AT₁R antagonism and PPARγ partial agonism, the three parts of telmisartan including the distal benzimidazole ring, the biphenyl moiety, and the carboxylic acid group experienced modification by core hopping method in our study. The central benzimidazole ring, however, remained intact considering its great affinity toward AT₁R and PPARγ. We utilized computational techniques for the sake of details on the binding interactions and conformational stability. Standard precision docking analysis and absorption, distribution, metabolism, excretion, and toxicity prediction received 10 molecules with higher Glide scores, similar interactions, and improved pharmacokinetic profiles compared to telmisartan. Comp#91 with highest scores for AT₁R (?11.92 kcal/mol) and PPARγ (?13.88 kcal/mol) exhibited excellent binding modes and pharmacokinetic parameters. Molecular dynamics trajectories on best docking pose of comp#91 confirmed the docking results and verified the conformational stability with both receptors throughout the course of 20-ns simulations. Thus, comp#91 could be identified as a promising lead in the development of dual AT₁R antagonist and PPARγ partial agonist against hypertension and type 2 diabetes.     

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Live cell measurements of interaction forces and binding kinetics between Discoidin Domain Receptor 1 (DDR1) and collagen I with atomic force microscopy

BackgroundDiscoidin Domain Receptors (DDRs) are membrane-tethered proteins of the receptor tyrosine kinase family, which signal in response to collagen. DDR expression is associated with human diseases, including fibrosis and cancer. The role of DDRs in human pathogenesis is mediated by dysregulated receptor function in response to the collagenous milieu. Thus, understanding DDR-collagen interactions is important for developing novel therapeutic strategies against DDRs.MethodsWe developed a biophysical method to isolate and measure specific interactions between DDR1 and collagen in live cells at the single molecule level using atomic force microscopy. This new method is capable of providing density and kinetics of membrane receptors in live cells.ResultsWe isolated DDR1-collagen interactions and quantified the association and dissociation rates of the DDR1-collagen I complex. We estimated separate binding probabilities of collagen I to DDR and integrin, and by combining kinetic and binding probability data, we were able to estimate the density of receptors in two cancer cell types. We also tested the viability of a DDR1 blocking antibody and determined its efficacy in suppressing DDR1-collagen binding.ConclusionsThe new method shows promise in quantifying receptor-ligand kinetics and receptor density on live cells.General significanceThe new approach is applicable to other receptor-ligand systems and allows the determination of critical parameters at the single cell/single molecule level – in particular, the direct determination of kinetic and density differences of receptors in different cell types. This capability should prove to be useful in cancer research and drug design.     

Reversible Inhibition of Cytosolic Glucocorticoid Receptors by Mercurial Agents. Application in the Measurement of Total and Unoccupied Receptors

Abstract

Recently developed “exchange assays” have been used to measure total cytosolic glucocorticoid receptor (GR) binding activity as compared to standard GR assays which measure unoccupied receptor. In the current study we modified these methods and extended the applications of such assays. Experiments defined the conditions whereby two sulfhydryl-binding agents, p-hydroxymercuribenzoate (PHMB) and mersalyl, completely inhibited binding of the glucocorticoid receptor to ligand in mouse renal cytosol. Reactivation of steroid-binding activity was restored by addition of dithiothreitol. The present study demonstrates 12% higher GR binding activity when this exchange assay is performed using saturated glucocorticoid-receptor complexes, rather than standard cytosol. Combining the data from the standard and exchange mouse renal cytosolic GR assays, it was determined that, at physiologic tissue corticosterone levels, the respective mean concentrations of unoccupied, occupied, and total GR were 467, 89, and 556 fmol/mg cytosol protein. Measurement of receptor concentrations by the use of these methods permits precise experimental differentiation of factors which affect total, as well as unoccupied GR.     

Agonist Binding to Mammalin Beta1 and Beta2 Adrenoceptors: Modulation by Guanine Nucleotides and Magnesium

Abstract

Agonist interaction with beta₁ and beta₂ adrenoceptors in rat rabbit lung has been examined using ligand binding techniques and the results are discussed in relation to current models of beta-adrenoceptor-adenylate cyclase coupling. Agonist binding has been assessed by examining the ability of isoprenaline or salbutamol to displace the labelled antagonist ³H-dihydroalprenolol (³H-DHA) from specific receptor sites. beta₁ and beta₂ adrenoceptors, even within the same tissue, exhibited different ion and temperature requirements for guanine nucleotide modulation of agonist binding. Thus, in contrast to the situation at beta₂ sites, agonist binding to beta₁ adrenoceptors was only sensitive to GTP if incubations were performed at physiological temperatures in the presence of Mg++ ions. These findings suggest that there may be different receptor-effector coupling relationships between the two beta-adrenoceptor subtypes.