A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

激动剂作用下α1-肾上腺素受体亚型在HEK293A细胞中的分布变化

为了明确α1-肾上腺素受体（α1-adrenergic receptor,α1-AR）三种亚型在人胚胎肾（human embryonic kidney,HEK）293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现：（1）α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90％以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数（配）相等;（2）在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中：（3）用α1-AR激动剂苯‘肾上腺素（phenylephrine,PE）刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR二种亚型在HEK293A细胞中的定位特点和分布变化各有不同。     

Transgenic studies of alpha(1)-adrenergic receptor subtype function

Mice with altered alpha(1)-adrenergic receptor (AR) genes have become important tools in elucidating the subtype-specific functions of the three alpha(1)-AR subtypes because of the lack of sufficiently subtype-selective pharmacological agents. Mice with a deletion (knockout, KO) or an overexpression (transgenic, TG) of the alpha(1A)-, alpha(1B)-, or alpha(1D)-AR subtypes have been generated. The alpha(1)-ARs are the principal mediators of the hypertensive response to alpha(1)-agonists in the cardiovascular system. Studies with these mice indicate that alpha(1A)-AR and alpha(1B)-AR subtypes play an important role in cardiac development and/or function as well as in blood pressure (BP) response to alpha(1)-agonists via vasoconstriction. The alpha(1B)- and alpha(1D)-subtypes also appear to be involved in central nervous system (CNS) processes such as nociceptive responses, modulation of memory consolidation and working memory. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from these studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes.     

High-throughput receptor-binding methods for somatostatin receptor 2

Three high-throughput screening methods for quantitating 125I-SS14 binding to human somatostatin receptor 2 (hSST2) have been developed. Microplate-based separation assays were performed in Packard Unifilter and Millipore Multiscreen plates. A homogeneous ligand-binding assay was developed by employing wheat germ agglutinin (WGA)-coated Flashplates. Apparent dissociation constants for 125I-SS14 binding to hSST2 were obtained with each method. IC(50) values were determined for 12 compounds using each of the methods. Similar IC(50) values were obtained for each compound with all of the methods. The WGA-Flashplate is suitable for fully automated high-throughput screening whereas the Unifilter and Multiscreen methods are more suitable for semiautomated and manual screening applications.     

Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.     

Differential coupling of beta3A- and beta3B-adrenergic receptors to endogenous and chimeric Galphas and Galphai

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G(alpha)q with the COOH-terminal heptapeptide of G(alpha)s or G(alpha)i were used to assess the relative coupling of beta(3)-adrenergic receptor (beta(3)-AR) splice variants (beta(3A) and beta(3B)) to G(alpha)s and G(alpha)i. The G(alpha)q/s and G(alpha)q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca(2+)(i)). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat beta(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta(3)-AR-induced coupling to G(alpha)q/s produced a rapid eightfold increase in Ca(2+)(i) followed by a slow decay to levels 25% above baseline. G(alpha)q/i also linked rat beta(3)-AR to mobilization of Ca(2+)(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca(2+)(i) was only 30% of the response obtained with G(alpha)q/s. Activation of the rat beta(3)-AR also increased GTP binding to endogenous G(alpha)i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse beta(3A)- and beta(3B)-AR to G(alpha)i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G(alpha)q/s and to increases in intracellular cAMP through endogenous G(alpha)s. The beta(3A)- and beta(3B)-AR coupled equivalently to G(alpha)q/i, but the temporal patterns of Ca(2+)(i) mobilization indicated that coupling was significantly less efficient than coupling to G(alpha)q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta(3A)- and beta(3B)-AR to G(alpha)i vs. G(alpha)s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta(3)-AR activation.     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

Identification of binding sites of prazosin,tamsulosin and KMD-3213 with alpha(1)-adrenergic receptor subtypes by molecular modeling

This investigation was performed to assess the importance of interaction in the bindings of selective and nonselective alpha(1)-antagonists to alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes using molecular modeling. The alpha(1)-antagonists used in this study were prazosin, tamsulosin and KMD-3213. Molecular modeling was performed on Octane 2 workstation (Silicon Graphics) using Discover/Insight II software (Molecular Simulations Inc.). Through molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains (TM) 3, 4, 5 and 6 of the alpha(1)-AR subtypes. In prazosin, the 4-amino group, 1-nitrogen atom and two methoxy groups of quinazoline ring possibly interact with the amino acids in TM3, TM5 and TM6 of alpha(1)-ARs. In tamsulosin, amine group of ethanyl amine chain, methoxy group of benzene ring and sulfonamide nitrogen of benzene ring interacts in TM3, TM4 and TM5 of alpha(1)-ARs. In KMD-3213, amine of ethyl amine chain and indoline nitrogen of this compound possibly interact within TM3 and TM5 of alpha(1)-ARs. Amide nitrogen of KMD-3213 also interacts within TM4 of alpha(1A)-AR. The results of the present study suggested that prazosin has similar binding sites in all the alpha(1)-AR subtypes while tamsulosin interacts at higher number of sites with alpha(1D)-subtype than other alpha(1)-AR subtypes. KMD-3213 being an alpha(1A)-AR selective ligand, binds to higher number of sites of alpha(1A) subtype than to other subtypes. All these amino acids are located near the extracellular loop. These findings are consistent with the previous studies that antagonists bind higher in the pocket closer to the extracellular surface unlike agonist binding.     

Functional studies with membrane-bound and detergent-solubilized alpha2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

A high-throughput fluorescence polarization assay for inhibitors of gyrase B

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.     

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.     

Three-dimensional models of alpha(2A)-adrenergic receptor complexes provide a structural explanation for ligand binding.

We have compared bacteriorhodopsin-based (alpha(2A)-AR(BR)) and rhodopsin-based (alpha(2A)-AR(R)) models of the human alpha(2A)-adrenengic receptor (alpha(2A)-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the alpha(2A)-AR(R) model provides a better explanation for ligand binding than does our alpha(2A)-AR(BR) model. Thus, we have made an extensive analysis of ligand binding to alpha(2A)-AR(R) and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the alpha(2A)-adrenergic receptor.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.     

New pyrimido[5,4-b]indoles and [1]benzothieno[3,2-d]pyrimidines: high affinity ligands for the alpha(1)-adrenoceptor subtypes

A number of new pyrimido[5,4-b]indole and [1]benzothieno[3,2-d]pyrimidine derivatives were synthesized and evaluated for their binding and functional properties at alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. They behaved as potent alpha(1)-AR antagonists. In binding experiments, some of them (RC24 and RC23) showed very high affinity for the alpha(1D)-AR subtype.     

A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.