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PubMed2004

Applicability of discovery science approach to determine biological effects of mobile phone radiation.

We argue that the use of high-throughput screening techniques, although expensive and laborious, is justified and necessary in studies that examine biological effects of mobile phone radiation. The "case of hsp27 protein" presented here suggests that even proteins with only modestly altered (by exposure to mobile phone radiation) expression and activity might have an impact on cell physiology. However, this short communication does not attempt to present the full scientific evidence that is far too large to be presented in a single article and that is being prepared for publication in three separate research articles. Examples of the experimental evidence presented here were designed to show the flow of experimental process demonstrating that the use of high-throughput screening techniques might help in rapid identification of the responding proteins. This, in turn, can help in speeding up of the process of determining whether these changes might affect human health.*

Cell Phone↗
PubMed2006

Discovery and characterization of inhibitors of human palmitoyl acyltransferases.

The covalent attachment of palmitate to specific proteins by the action of palmitoyl acyltransferases (PAT) plays critical roles in the biological activities of several oncoproteins. Two PAT activities are expressed by human cells: type 1 PATs that modify the farnesyl-dependent palmitoylation motif found in H- and N-Ras, and type 2 PATs that modify the myristoyl-dependent palmitoylation motif found in the Src family of tyrosine kinases. We have previously shown that the type 1 PAT HIP14 causes cellular transformation. In the current study, we show that mRNA encoding HIP14 is up-regulated in a number of types of human tumors. To assess the potential of HIP14 and other PATs as targets for new anticancer drugs, we developed three cell-based assays suitable for high-throughput screening to identify inhibitors of these enzymes. Using these screens, five chemotypes, with activity toward either type 1 or type 2 PAT activity, were identified. The activity of the hits were confirmed using assays that quantify the in vitro inhibition of PAT activity, as well as a cell-based assay that determines the abilities of the compounds to prevent the localization of palmitoylated green fluorescent proteins to the plasma membrane. Representative compounds from each chemotype showed broad antiproliferative activity toward a panel of human tumor cell lines and inhibited the growth of tumors in vivo. Together, these data show that PATs, and HIP14 in particular, are interesting new targets for anticancer compounds, and that small molecules with such activity can be identified by high-throughput screening.

Acyltransferases↗
PubMed2002

Development of a green fluorescent protein-based cell bioassay for the rapid and inexpensive detection and characterization of ah receptor agonists.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals. Although halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs, respectively) represent the highest affinity and most toxic ligands, recent studies have demonstrated that the AhR can be activated by chemicals with structures distinctly different from HAHs/PAHs. In order to identify and characterize novel AhR ligands, we developed a rapid and inexpensive high-throughput screening bioassay based on the ability of AhR agonists to induce an HAH/PAH-responsive, enhanced green fluorescent protein (EGFP) reporter gene in a stably transfected mouse hepatoma (Hepa1c1c7) cell line. EGFP induction in the resulting recombinant cell line, H1G1.1c3, is sensitive (with a minimal 1-pM detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin, the most potent AhR ligand), and it responds to HAHs and PAHs in a time-, dose-, and chemical-specific manner. Application of this bioassay was demonstrated by the rapid characterization of the relative inducing potency of a series of previously uncharacterized dioxin surrogates. This bioassay system has numerous advantages over currently available AhR-based bioassays including increased rapidity and ease of use, low reagent cost, and application for high-throughput screening.

Animals↗
PubMed2001

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.

Amidohydrolases↗
PubMed2005

High-throughput RNA interference screens in Drosophila tissue culture cells.

This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.

Animals↗
PubMed1998

An enzyme-linked immunosorbent assay for the detection of agents which interfere with the DNA binding activities of transcription factors--exemplified by NF-IL6.

Binding of transcription factors to DNA is usually detected by the electrophoretic mobility shift assay also known as gel-shift or band-shift assay. Recently the use of biosensors has allowed factor/DNA interactions to be followed in real time. However, neither of these approaches lends itself easily to high-throughput screening of agents which might interfere with this process. We have therefore developed a 96-well plate-based enzyme-linked immunosorbent assay for this purpose. Biotinylated oligonucleotides bound to streptavidin-coated plates are used to capture recombinant transcription factor proteins which can then be detected using specific antibodies which in turn are recognised by peroxidase-conjugated antisera. The peroxidase catalyzes conversion of a colorless substrate to a colored product which can be quantified by optical densitometry on a plate reader. Agents which interfere with the binding of the transcription factor to DNA reduce the optical density in the well. Using the NF-IL6 transcription factor we show that the assay can detect reductions to 86% at significant levels. The assay may be readily adapted for robotic manipulation, making it ideal for high-throughput screening.

Base Sequence↗
PubMed2002

Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.

Antineoplastic Agents↗
PubMed2006

Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution.

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.

Binding Sites↗
PubMed2002

An Escherichia coli biosensor strain for amplified and high throughput detection of antimicrobial agents.

We report here the construction of a bacterial reporter system for high-throughput screening of antimicrobial agents. The test organism is the Escherichia coli K-12 strain carrying luciferase genes luxC, luxD, luxA, luxB, and luxE from the bioluminescent bacterium Photorhabdus luminescens in a runaway replication plasmid. The replication of the plasmid can be induced, resulting in a change of the plasmid copy number from 1-2/cell to several hundreds per cell within tens of minutes. This increase in plasmid copies is independent of the replication of the host cells. The system will therefore amplify the effects of antibiotics inhibiting bacterial replication machinery, such as fluoroquinolones, and the inhibitory effects can be measured in real time by luminometry. The biosensor was compared with a strain engineered to emit light constitutively, and it was shown to be much more sensitive to various antibiotics than conventional overnight cultivation methods. The approach shows great potential for high-throughput screening of new compounds.

Anti-Bacterial Agents↗
PubMed2005

Cell-based microarrays: current progress, future prospects.

Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a novel method for performing high-throughput screens of gene function. In this study, expression vectors containing the open reading frame of human genes were printed onto glass microscope slides to form a microarray. Transfection reagents were added pre- or post-spotting, and cells grown over the surface of the array. They demonstrated that cells growing in the immediate vicinity of the expression vectors underwent 'reverse transfection', and that subsequent alterations in cell function could then be detected by secondary assays performed on the array. Subsequent publications have adapted the technique to a variety of applications, and have also shown that the approach works when arrays are fabricated using short interfering RNAs and compounds. The potential of this method for performing analyses of gene function and for identifying novel therapeutic agents has been clearly demonstrated, and current efforts are focused on improving and harnessing this technology for high-throughput screening applications.

Animals↗
PubMed2000

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly.

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.

Apolipoproteins↗
PubMed2005

High-throughput enzyme assay on a multichannel microchip using optically gated sample introduction.

To meet the requirements for high-throughput screening for drug discovery research, it is very important to develop techniques with the ability of performing multiple enzyme assays simultaneously. Using optically gated sample introduction on a multichannel microchip, multiple enzyme assays have been demonstrated in four parallel channels. The hydrolysis of fluorescein mono-beta-D-galactopyranoside by beta-galactosidase and the inhibition of this reaction by the competitive inhibitor phenylethyl beta-D-thiogalactoside were initially studied to determine the effect of system movement using the voice coil actuator on the enzyme assay reaction. The results from these two studies are consistent with the results from the assay using a single-channel microchip, and they demonstrate that the system using optically gated sample introduction on multichannel microchip can be used to perform multiple enzyme assays. Three unique enzyme assays were also performed in different channels, which show this technique could be competitive for high-throughput screening in drug discovery with other traditional techniques.

Animals↗
PubMed2006

A homogeneous scintillation proximity assay for acetyl coenzyme A carboxylase coupled to fatty acid synthase.

We have devised a rapid and sensitive homogeneous assay for acetyl CoA carboxylase (ACC) in a scintillation proximity assay format suitable for high-throughput screening. In this assay, ACC is coupled to fatty acid synthase (FAS). Malonyl CoA, the product of the ACC reaction, and acetyl CoA serve as substrates for FAS to synthesize palmitic acid. When [(3)H]acetyl CoA is used in the ACC/FAS coupled system, [(3)H]palmitic acid, the final product, is readily detected by scintillation proximity in a FlashPlate or Image FlashPlate coated with phospholipid. The [(3)H]palmitic acid binds to the coated phospholipid through hydrophobic interaction which brings it into close proximity of the scintillant on the FlashPlate or the Image FlashPlate, yielding photons that are read in a TopCount or LeadSeeker, respectively. The current assay consists of simple reagent addition, incubation, and detection of signal. The signal is approximately 30-fold over the background and the Z' value is approximately 0.80, suggesting that this assay is robust and highly reproducible. To our knowledge this ACC/FAS coupled scintillation proximity assay is the only assay format that is compatible with high-throughput screening for systematic search of inhibitors against mammalian ACC.

Acetyl-CoA Carboxylase↗
PubMed2004

Potentiometric quasi-array employing calixarene derivatives for the high-throughput similarity/diversity screening of beta-adrenergic and beta-blocking chiral drugs by HPLC.

Performance of potentiometric quasi-array detection system consisted with the seven poly (vinyl chloride) (PVC) based liquid membrane electrodes in the cation-exchange HPLC using acetonitrile--40 mM phosphoric acid (15 : 85, v/v, pH* 2.35) for assessing of molecular similarity/diversity in a mini-library of beta-adrenergic and beta-blocking chiral drugs was presented. Macrocyclic compounds differing in stability of their conformers as well as in a size, steric hindrance and polarity of its internal cavities, comprising a series of five calix[6]arene derivatives completed with one modified calix[4]resorcinarene, were used as neutral ionophores to compose mentioned set of PVC-based electrodes. The output potentiometric responses were registered in the linear supernerstian range of calibration graph of each electrode, i.e. for a constant injected concentration 2.0 x 10(-4) M of investigated drugs, which is related to the amount frequently used at in vitro studies on pharmacological effects of these drugs. The impact of symmetry oriented supramolecular interactions on the responses of developed electrodes were characterised with proposed series of the highly significant quantitative structure-potentiometric response relationships (QSPRRs) combining both three-dimensional (3D) molecular descriptors of analysed drugs as well as lipophilicity and volume polarizability of calixarene-type ionophores. The principal components analysis (PCA) and unweighted hierarchical clustering analysis (HCA) were used as the pattern recognition techniques into collected potentiometric database for extraction of the useful information on the molecular and pharmacological similarity/diversity of analysed drugs, thus a high-throughput and consistent identification of therapeutically relevant agonists of beta2- and beta3-adrenoceptors and antagonists of beta1-adrenoceptor was especially achieved. This evidence supports also a hypothesis formulated by results of homology modelling on the subtle significance of an unrecognised supramolecular insertion processes of the chiral drug molecule into the flexible hydrophobic pocket(s) formed by seven helical transmembrane moving domains of beta-adrenoceptors on their final activation, sequestration, down-regulation or blockade.

Adrenergic beta-Antagonists↗
PubMed2005

Development of a high-throughput in vitro assay using a novel Caco-2/rat hepatocyte system for the prediction of oral plasma area under the concentration versus time curve (AUC) in rats.

INTRODUCTION: Previously, we have shown that a novel Caco-2/human hepatocyte system is a useful model for the prediction of oral bioavailability in humans. In this study, we attempted to use a similar system in a high-throughput screening mode for the selection of new compound entities (NCE) in drug discovery. METHODS: A total of 72 compounds randomly selected from three different chemotypes were dosed orally in rats. In vivo plasma area under the concentration versus time curve (AUC) from 0-6 h of the parent compound was determined. The same compounds were also tested in the Caco-2/rat hepatocyte system. In vitro AUC from 0-3 h in the Caco-2 rat hepatocyte system was determined. RESULTS: The predictive usefulness of the Caco-2/rat hepatocyte system was evaluated by comparing the in vivo plasma AUC and the in vitro AUC. Linear regression analysis showed a reasonable correlation (R2 = 0.5) between the in vivo AUC and the in vitro AUC. Using 0.4 microM h in vivo AUC as a cut-off, compounds were categorized as either low or high AUC. The in vitro AUC successfully matched the corresponding in vivo category for sixty-three out of seventy-two compounds. DISCUSSION: The results presented in this study suggest that the Caco-2/rat hepatocyte system may be used as a high-throughput screen in drug discovery for pharmacokinetic behaviors of compounds in rats.

Administration, Oral↗
PubMed2003

Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening.

Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.

Amino Acid Sequence↗
PubMed2004

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.

Chromatography, Liquid↗
PubMed2001

Development of a fluorescence based high throughput assay for antagonists of the human chorionic gonadotropin receptor extracellular domain: analysis of peptide inhibitors.

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

Automation↗