Small Molecules Revealed in a Screen Targeting Epithelial Scattering Are Inhibitors of Microtubule Polymerization
Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in the detachment of cell-cell junctions and initiation of cell migration. Instead of coordinating collective cell behavior within a tissue, cells become solitary and have few cell-cell interactions. Since epithelial scattering is recapitulated in cancer progression and since HGF signaling drives cancer metastasis in many cases, inhibitors of HGF signaling have been proposed to act as anticancer agents. We previously sought to better understand critical components required for HGF-induced epithelial scattering by performing ...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Hoj, T. H., Robinson, R. J., Burton, J. C., Densley-Ure, R. A., Olson, T. V., Williams, L. K., Coward, L., Gorman, G., Hansen, M. D. H. Tags: Original Research Source Type: research
Novel Biomarkers for Renal Diseases? None for the Moment (but One)
Recent years have witnessed the unprecedented development and integration of genomics, epigenetics, transcriptomics, proteomics, and metabolomics, as well as a growing interest in novel single biomarkers and process-specific biomarker panels in human renal diseases. In a scenario currently dominated by kidney biopsy and established biomarkers such as serum creatinine, albuminuria, and proteinuria, novel biomarkers could potentially provide vital diagnostic and prognostic information and help to predict response to treatment in several clinical settings, including acute kidney injury, renal transplant, autosomal dominant po...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Gentile, G., Remuzzi, G. Tags: Review Source Type: research
Corrigendum
Pedró-Rosa, L.; Buckner, F.; Ranade, R. M.; et al. Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening. J Biomol Screen. 2015, 20, 122–130. (Original DOI: 10.1177/1087057114548832) (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Tags: Other Source Type: research
Identification of HDAC Inhibitors Using a Cell-Based HDAC I/II Assay
In this study, we describe a screening approach for identification of compounds that inhibit endogenous class I and II HDACs. A homogeneous, luminogenic HDAC I/II assay was optimized in a 1536-well plate format in several human cancer cell lines, including HCT116 and human neural stem cells. The assay confirmed 37 known HDAC inhibitors from two libraries of known epigenetics-active compounds. Using the assay, we identified a group of potential HDAC inhibitors by screening the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of 2527 small-molecule drugs. The selected compounds showed si...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Hsu, C.-W., Shou, D., Huang, R., Khuc, T., Dai, S., Zheng, W., Klumpp-Thomas, C., Xia, M. Tags: Technical Note Source Type: research
Small-Activating RNA Can Change Nucleosome Positioning in Human Fibroblasts
In this study, we examined changes in nucleosome repositioning and the involvement of RNA polymerase II (RNAPII) in this process. We screened saRNAs for OCT4 (POU5F1), SOX2, and NANOG, and identified several novel saRNAs. We found that nucleosome positioning was altered after saRNA treatment and that the formation of nucleosome-depleted regions (NDRs) contributed to RNAa at sites of RNAPII binding, such as the TATA box, CpG islands (CGIs), proximal enhancers, and proximal promoters. Moreover, RNAPII appeared to be bound specifically to NDRs. These results suggested that changes in nucleosome positions resulted from RNAa. W...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Wang, B., Sun, J., Shi, J., Guo, Q., Tong, X., Zhang, J., Hu, N., Hu, Y. Tags: Original Research Source Type: research
A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway
Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated ...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Voter, A. F., Manthei, K. A., Keck, J. L. Tags: Original Research Source Type: research
A Comparison of LC-MS/MS and a Fully Integrated Autosampler/Solid-Phase Extraction System for the Analysis of Protein Binding Samples
A new analysis approach was evaluated for measuring plasma protein binding (PPB) of small molecules using the Agilent RapidFire high-throughput system coupled with a Sciex API 4000 mass spectrometer (RF-MS/MS). Thirty-three proprietary and 12 literature compounds were subjected to rapid equilibrium dialysis (RED) and evaluated in parallel using RF-MS/MS at 16.4 s/sample and traditional liquid chromatography–tandem mass spectrometry (LC-MS/MS) at 3.5 min/sample, thus making the RF-MS/MS analysis over 12 times faster than LC-MS/MS. The high-throughput analysis method that was developed demonstrated excellent correlatio...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Amaral, A., Saran, C., Amin, J., Hatsis, P. Tags: Original Research Source Type: research
Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-{kappa}B Pathway
The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-B pathway model system were identified by integrating affinity selection–mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-B proteins in parallel against large-compound libraries. ALIS identified 382 t...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Kutilek, V. D., Andrews, C. L., Richards, M. P., Xu, Z., Sun, T., Chen, Y., Hashke, A., Smotrov, N., Fernandez, R., Nickbarg, E. B., Chamberlin, C., Sauvagnat, B., Curran, P. J., Boinay, R., Saradjian, P., Allen, S. J., Byrne, N., Elsen, N. L., Ford, R. E Tags: Original Research Source Type: research
Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction
In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni2+-...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Brenke, J. K., Salmina, E. S., Ringelstetter, L., Dornauer, S., Kuzikov, M., Rothenaigner, I., Schorpp, K., Giehler, F., Gopalakrishnan, J., Kieser, A., Gul, S., Tetko, I. V., Hadian, K. Tags: Original Research Source Type: research
Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity
The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Pinto, A. F., Ebrahimi, M., Saleeb, M., Forsberg, A., Elofsson, M., Schüler, H. Tags: Original Research Source Type: research
NMR Binding and Functional Assays for Detecting Inhibitors of S. aureus MnaA
Nonessential enzymes in the staphylococcal wall teichoic acid (WTA) pathway serve as highly validated β-lactam potentiation targets. MnaA (UDP-GlcNAc 2-epimerase) plays an important role in an early step of WTA biosynthesis by providing an activated form of ManNAc. Identification of a selective MnaA inhibitor would provide a tool to interrogate the contribution of the MnaA enzyme in the WTA pathway as well as serve as an adjuvant to restore β-lactam activity against methicillin-resistant Staphylococcus aureus (MRSA). However, development of an epimerase functional assay can be challenging since both MnaA substrat...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Hou, Y., Mayhood, T., Sheth, P., Tan, C. M., Labroli, M., Su, J., Wyss, D. F., Roemer, T., McCoy, M. A. Tags: Original Research Source Type: research
High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries
It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegene...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Perez del Palacio, J., Diaz, C., de la Cruz, M., Annang, F., Martin, J., Perez-Victoria, I., Gonzalez-Menendez, V., de Pedro, N., Tormo, J. R., Algieri, F., Rodriguez-Nogales, A., Rodriguez-Cabezas, M. E., Reyes, F., Genilloud, O., Vicente, F., Galvez, J. Tags: Original Research Source Type: research
Development of an Enhanced Phenotypic Screen of Cytotoxic T-Lymphocyte Lytic Granule Exocytosis Suitable for Use with Synthetic Compound and Natural Product Collections
We previously developed an assay of cytotoxic T-lymphocyte lytic granule exocytosis based on externalization of LAMP-1/CD107A using nonphysiological stimuli to generate maximal levels of exocytosis. Here, we used polystyrene beads coated with anti-CD3 antibodies to stimulate cells. Light scatter let us distinguish cells that contacted beads from cells that had not, allowing comparison of signaling events and exocytosis from stimulated and unstimulated cells in one sample. Bead stimulation resulted in submaximal exocytosis, making it possible to detect compounds that either augment or inhibit lytic granule exocytosis. Coupl...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Zhao, Z., deMayo, J. A., West, A. M., Balunas, M. J., Zweifach, A. Tags: Original Research Source Type: research
384-Well Multiplexed Luminex Cytokine Assays for Lead Optimization
Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturi...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Tang, H., Panemangalore, R., Yarde, M., Zhang, L., Cvijic, M. E. Tags: Original Research Source Type: research
Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening
Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term tr...
Source: Journal of Biomolecular Screening - June 20, 2016 Category: Molecular Biology Authors: Doucette, J., Zhao, Z., Geyer, R. J., Barra, M. M., Balunas, M. J., Zweifach, A. Tags: Original Research Source Type: research
