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PubMed2005

High throughput screening for protein kinase inhibitors.

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of pathologies. Since the discovery that the v-Src oncogene encoded a protein kinase in 1978, kinases have remained a focus of research for pharmaceutical laboratories and academic groups alike. Many have sought to develop orally available low molecular weight synthetic kinase modulators (predominantly inhibitors) and thus capitalize on the links between aberrant regulation and disease. This interest in kinases as drug targets was fueled in recent years by the success of several kinase inhibitors in the clinic, primarily Gleevec for the treatment of chronic myelogenous leukemia and Iressa for the treatment of advanced non-small cell lung cancer. This review focuses on the development of small molecule drugs, most of them binding in or close to the ATP binding pocket. After some general considerations regarding the selection of a particular kinase for drug discovery, we will discuss the encouraging lessons learned from some of the kinase inhibitors currently in various stages of development. The majority of this review is dedicated to a detailed description and discussion of the various assay formats currently being employed for high throughput screening.

Animals↗
PubMed2000

A novel high throughput screening assay for HCV NS3 helicase activity.

A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate technology. This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5' termini. This DNA duplex was immobilized, via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate (NEN Life Science Products). Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells. Biochemical characterization of the HCV NS3 helicase activity was performed using this assay. We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction. Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation. As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl(2), MnCl(2), KCl, EDTA, and by pH. We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate. Therefore, this Flashplate helicase assay is fast, convenient and reproducible. These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors.

Animals↗
PubMed2001

Solid-supported lipid membranes as a tool for determination of membrane affinity: high-throughput screening of a physicochemical parameter.

Quantification of membrane affinity is an important early screening step in modern drug design. However, current approaches using different lipid membrane models usually are time-consuming or show severe experimental drawbacks. In this paper we describe the use of solid-supported lipid membranes (TRANSIL) as a new tool for the determination of membrane affinity. Eighteen pharmaceuticals (neutrals, acids, and bases) have been analyzed for their lipophilicity at physiological pH in an automated setup; phase separation of lipid and aqueous phase can be achieved simply by a short low-speed centrifugation or filtration. The membrane affinity is then calculated by quantification of the total drug concentration and the amount of drug remaining in the aqueous phase after incubation with solid-supported lipid membranes. Lipophilicity parameters relying on solid-supported lipid membranes correlate well with octanol-water partition coefficients K(ow) for neutral organic compounds (range of log K(ow) = 1.5-5, n = 7, r = 0.93). Data acquisition with this lipid membrane model system is highly re-producible. Even in the case of ionizable drugs, where K(ow) tends to underestimate membrane affinity, the latter can be correctly quantified using solid-supported lipid membranes: data comparison shows good agreement of the presented approach with established but time-consuming standardized lipid/buffer systems. Solid-supported lipid membranes allow a fast and reliable quantification of membrane affinity, enabling high-throughput screening of this physicochemical parameter.

Drug Design↗
PubMed2005

Determining the optimal size of small molecule mixtures for high throughput NMR screening.

High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a "hit" for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library.

Chemistry, Pharmaceutical↗
PubMed2003

Development of a cell-based enzyme-linked immunosorbent assay for high-throughput screening of HIV type 1 entry inhibitors targeting the coreceptor CXCR4.

CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1) X4 virus, plays an important role in virus entry into the target cells. Antiviral agents that bind to CXCR4 are expected to inhibit HIV-1 entry. A cell-based enzyme-linked immunosorbent assay (ELISA) was developed utilizing an anti-CXCR4 monoclonal antibody, 12G5, and cells expressing CD4 and CXCR4, U373-MAGI-CXCR4(CEM) cells. Using this assay, we demonstrated that 12G5 specifically binds to the CXCR4-expressing cells, but not to CCR5-expressing cells and cells without CXCR4 and CCR5, consistent with the results obtained by using flow cytometry. The well-characterized CXCR4 antagonists, T22, T14012 (a downsized analog of T-22), and AMD3100, effectively inhibited 12G5 binding to CXCR4-expressing cells, while HIV-1 entry inhibitors targeting CD4 and gp41 as well as HIV-1 reverse transcriptase and protease inhibitors did not block the binding of 12G5 to U373-MAGI-CXCR4(CEM) cells. The prepared plates containing the fixed cells could be stored at -80 degrees C for at least 5 months without affecting the cell reactivity with 12G5, which enhances the convenience of this method. This suggests that the cell-based ELISA is specific, sensitive, convenient, rapid, and economic. With a robotic sample processing system, this assay can be used for high-throughput screening of HIV-1 entry inhibitors targeted to the HIV-1 coreceptor CXCR4.

Anti-HIV Agents↗
PubMed2006

Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma.

BACKGROUND: Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. METHODS: We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. RESULTS: We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. CONCLUSIONS: The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.

Antibodies, Monoclonal↗
PubMed2004

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Acridines↗
PubMed2004

High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein.

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.

Animals↗
PubMed2004

High throughput screening and characterization of HIV-1 entry inhibitors targeting gp41: theories and techniques.

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays an important role in HIV-1 entry and severs as an attractive target for development of HIV-1 entry inhibitors, a new class of anti-HIV drugs. Triggered by gp120 binding to CD4 and a coreceptor, gp41 undergoes a conformation shift from a native prefusogenic state to a fusogenic state, in which the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) associate to form a six-helix bundle, representing the fusion-active gp41 core. Any compound that disrupts the gp41 six-helix bundle formation may inhibit the gp41-mediated membrane fusion, thereby blocking HIV-1 entry into target cells. Peptides derived from the gp41 NHR and CHR regions, designated N- and C-peptides, can interact with the counterpart regions in gp41 and interfere with the six-helix bundle formation between the viral NHR and CHR region, thus inhibiting fusion of the virus with the target cell. One of the C-peptides, T-20 (brand name: Fuzeon), was recently approved by the US FDA as the first HIV entry inhibitor which can be used for treatment of AIDS patients who fail to respond to the current antiretroviral drugs, e.g., the reverse transcriptase inhibitors and protease inhibitors. The limitations of T-20 include lack of oral availability and high cost of production. Thus it is essential to develop small molecule HIV-1 entry inhibitors targeting gp41. This review summarizes the newly developed techniques for high throughput screening (HTS) and characterization of the HIV-1 entry inhibitors targeting gp41. The theories behind these techniques are also discussed. It is expected that the "drug-like" compounds with potent HIV-1 fusion inhibitory activity will be identified in the near future and used as leads for development of the low molecular weight HIV-1 entry inhibitors for the chemotherapy of HIV-1 infection and AIDS.

Animals↗
PubMed2000

Fluorescence polarization assays for high throughput screening of G protein-coupled receptors.

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to [(125)I]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 microM. These attributes suggest that accurate assessment of drug binding can be obtained.

Fluorescence Polarization↗
PubMed2003

TIP, a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

Adjuvants, Immunologic↗
PubMed2005

Evaluating the high-throughput screening computations.

The judges evaluated the submissions for the McMaster University High-Throughput Data-Mining and Docking Competition based on 3 criteria: identification of active compounds, percent enrichment, and overview of the competition. Using these metrics, 4 of the participating groups found meaningful enrichment, and 3 groups made perceptive comments about the general nature of the competition.

Computational Biology↗
PubMed2001

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.

We have previously demonstrated that Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery. To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by precoating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded. The complexes with a nanoparticle form were efficiently transduced into cells without use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.

Adenoviridae↗
PubMed2006

An optimal pooling strategy applied to high-throughput screening for rare marker-free transformants.

An optimal pooling system, called Accurate and Fast Target Screening, has been developed for high-throughput identifying the rare marker-free transformants. This system can identify targets between 10- and 100-fold more efficiently than analysis of individual samples. By calculating the efficiency for different proportions of targets and the optimal group size in a worst case scenario, we are able to estimate an upper limit for the number of tests that are required. The application of this system to determine the transgene in an artificially constructed population of transgenic and non-transgenic wheat lines successfully identified the 10 positive samples located randomly with 990 negative samples using only 92 PCR reactions. The same approach was also applied to determine transgene expression by SDS-PAGE of seed proteins. This system gives unambiguous positive or negative results and should facilitate marker-free transformation.

Algorithms↗
PubMed2001

A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30 microM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Automation↗
PubMed2006

A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Acetyltransferases↗
PubMed2006

Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

Aldose-Ketose Isomerases↗
PubMed1997

Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Anti-Bacterial Agents↗