Insights into PARP Inhibitors' Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays
In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD+ binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicabl...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Papeo, G., Avanzi, N., Bettoni, S., Leone, A., Paolucci, M., Perego, R., Quartieri, F., Riccardi-Sirtori, F., Thieffine, S., Montagnoli, A., Lupi, R. Tags: Technical Notes Source Type: research
Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins
Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptoti...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Hassig, C. A., Zeng, F.-Y., Kung, P., Kiankarimi, M., Kim, S., Diaz, P. W., Zhai, D., Welsh, K., Morshedian, S., Su, Y., O'Keefe, B., Newman, D. J., Rusman, Y., Kaur, H., Salomon, C. E., Brown, S. G., Baire, B., Michel, A. R., Hoye, T. R., Francis, S., Ge Tags: Original Research Source Type: research
Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer
Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire–mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes w...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Brooks, E., Wu, X., Hanel, A., Nguyen, S., Wang, J., Zhang, J. H., Harrison, A., Zhang, W. Tags: Original Research Source Type: research
Profiling of Nutrient Transporter Expression in Human Stem Cell-Derived Cardiomyocytes Exposed to Tyrosine Kinase Inhibitor Anticancer Drugs Using RBD Ligands
We applied a novel profiling approach using receptor binding domain (RBD) ligands to cell surface domains of a panel of nutrient transporters to characterize the impact of a number of tyrosine kinase inhibitor anticancer drugs on human stem cell–derived cardiomyocytes. High-content screening and flow cytometry analysis showed diagnostic changes in nutrient transporter expression correlating with glycolysis and oxidative phosphorylation–based cell metabolism in glucose and galactose media. Cluster analysis of RBD binding signatures of drug-treated cells cultured in glucose medium showed good correlation with sen...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Li, J., Cousin, C., Tinkler, H., Touhami, J., Petit, V., Thomas, N. Tags: Original Research Source Type: research
Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors
The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors—namely, N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)—are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain r...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Fukushima, K., Tabata, Y., Imaizumi, Y., Kohmura, N., Sugawara, M., Sawada, K., Yamazaki, K., Ito, M. Tags: Original Research Source Type: research
Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy
Niemann–Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells an...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Yu, D., Swaroop, M., Wang, M., Baxa, U., Yang, R., Yan, Y., Coksaygan, T., DeTolla, L., Marugan, J. J., Austin, C. P., McKew, J. C., Gong, D.-W., Zheng, W. Tags: Original Research Source Type: research
Development of Basal-Like HaCaT Keratinocytes Containing the Genome of Human Papillomavirus (HPV) Type 11 for Screening of Anti-HPV Effects
In conclusion, the established HPV11.HaCaT cells can provide us with a convenient and relatively stable tool for screening anti–HPV-11 agents. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Wang, Y., Li, X., Song, S., Wu, J. Tags: Original Research Source Type: research
Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors
RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5' end with fluorescein (FL...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Falk, S. P., Weisblum, B. Tags: Original Research Source Type: research
Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening
A novel, ultrahigh-throughput, fluorescence anisotropy–based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNAIle lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to elim...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Shapiro, A. B., Plant, H., Walsh, J., Sylvester, M., Hu, J., Gao, N., Livchak, S., Tentarelli, S., Thresher, J. Tags: Original Research Source Type: research
Screening Robotics and Automation
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: McGee, J. Tags: Product Focus Source Type: research
Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs
We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low tem...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Green, D. J., Rudd, E. A., Laugharn, J. A. Tags: Original Research Source Type: research
Virtual Screening of Some Active Human Macrophage Migration Inhibitory Factor Antagonists
Macrophage migration inhibitory factor (MIF) is an autocrine- and paracrine-acting cytokine that is involved in several inflammatory, autoimmune, infectious, and oncogenic diseases. Clinical data have shown that inhibition of MIF, especially its tautomerase activity, with small compounds has been beneficial in some disease models. A virtual screening (VS) experiment is conducted for searching some active compounds to inhibit the tautomerase activity of MIF from the ZINC database. By using an x-ray–determined structure OXIM-11 as the query and an in-house developed two-dimensional scaffold comparing method designated ...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Tsai, T.-L., Lin, T.-H. Tags: Original Research Source Type: research
Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening
The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a ...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Spicer, T., Fernandez-Vega, V., Chase, P., Scampavia, L., To, J., Dalton, J. P., Da Silva, F. L., Skinner-Adams, T. S., Gardiner, D. L., Trenholme, K. R., Brown, C. L., Ghosh, P., Porubsky, P., Wang, J. L., Whipple, D. A., Schoenen, F. J., Hodder, P. Tags: Original Research Source Type: research
High Specific DNAzyme-Aptamer Sensor for Salmonella paratyphi A Using Single-Walled Nanotubes-Based Dual Fluorescence-Spectrophotometric Methods
In this work, single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to target the flagellin protein. The selected aptamers were confirmed to have high sensitivity and specificity to the flagellin. In addition, a probe (P0) containing the DNAzyme and fluorescein isothiocyanate–labeled aptamer3 sequences was employed as a dual probe for observing fluorescence and absorbance changes. The flagellin demonstrated low detection limits of 5 ng/mL by fluorescence...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Ning, Y., Li, W., Duan, Y., Yang, M., Deng, L. Tags: Original Research Source Type: research
Identification of Novel Polyphenolic Inhibitors of Shikimate Dehydrogenase (AroE)
Shikimate dehydrogenase (AroE) is an attractive target for herbicides and antimicrobial agents due to its conserved and essential nature in plants, fungi, and bacteria. Here, we have performed an in vitro screen using a collection of more than 5500 compounds and identified 24 novel inhibitors of AroE from Pseudomonas putida. The IC50 values for the two most potent inhibitors we identified, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), were 3.0 ± 0.2 µM and 3.7 ± 0.5 µM, respectively. Based on the high level of structural conservation between AroE orthologs, we predicted that the i...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Peek, J., Shi, T., Christendat, D. Tags: Original Research Source Type: research
