Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast
In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach...
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Zhang, Y., Tian, D., Matsuyama, H., Hamazaki, T., Shiratsuchi, T., Terada, N., Hook, D. J., Walters, M. A., Georg, G. I., Hawkinson, J. E. Tags: Original Research Source Type: research
Fluorescence-Based Automated Screening Assay for the Study of the pH-Sensitive Channel ASIC1a
Acid-sensing ion channel 1a (ASIC1a) is involved in several pathologies, including neurodegenerative and neuroinflammatory disorders, stroke, epilepsy, and inflammatory pain. ASIC1a has been the subject of intense drug discovery programs devoted to the development of new pharmacological tools for its modulation. However, these efforts to generate new compounds have faced the lack of an efficient screening procedure. In the past decades, improvements in screening technologies and fluorescent sensors for the study of ion channels have provided new opportunities in this field. Unfortunately, ASIC1a is mainly a Na+ permeable c...
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Mazzocchi, N., De Ceglia, R., Mazza, D., Forti, L., Muzio, L., Menegon, A. Tags: Original Research Source Type: research
Focused Screening Identifies Evoxine as a Small Molecule That Counteracts CO2-Induced Immune Suppression
Patients with severe lung disease may develop hypercapnia, elevation of the levels of CO2 in the lungs and blood, which is associated with increased risk of death, often from infection. To identify compounds that ameliorate the adverse effects of hypercapnia, we performed a focused screen of 8832 compounds using a CO2-responsive luciferase reporter in Drosophila S2* cells. We found that evoxine, a plant alkaloid, counteracts the CO2-induced transcriptional suppression of antimicrobial peptides in S2* cells. Strikingly, evoxine also inhibits hypercapnic suppression of interleukin-6 and the chemokine CCL2 expression in human...
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Helenius, I. T., Nair, A., Bittar, H. E. T., Sznajder, J. I., Sporn, P. H. S., Beitel, G. J. Tags: Original Research Source Type: research
High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection
In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Islam, M. K., Baudin, M., Eriksson, J., Öberg, C., Habjan, M., Weber, F., Överby, A. K., Ahlm, C., Evander, M. Tags: Original Research Source Type: research
Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus
Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM....
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Sharma, R., Lambu, M. R., Jamwal, U., Rani, C., Chib, R., Wazir, P., Mukherjee, D., Chaubey, A., Khan, I. A. Tags: Original Research Source Type: research
A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension
In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor–β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibi...
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Cheng, H., Chen, T., Tor, M., Park, D., Zhou, Q., Huang, J. B., Khatib, N., Rong, L., Zhou, G. Tags: Original Research Source Type: research
An AlphaScreen Assay for the Discovery of Synthetic Chemical Inhibitors of Glucagon Production
Glucose homeostasis is primarily controlled by two opposing hormones, insulin and glucagon, and diabetes results when insulin fails to inhibit glucagon action. Recent efforts to control glucagon in diabetes have focused on antagonizing the glucagon receptor, which is effective in lowering blood glucose levels but leads to hyperglucogonemia in rodents. An alternative strategy would be to control glucagon production with small molecules. In pursuit of this goal, we developed a homogeneous AlphaScreen assay for measuring glucagon in cell culture media and used this in a high-throughput screen to discover synthetic compounds t...
Source: Journal of Biomolecular Screening - March 18, 2016 Category: Molecular Biology Authors: Evans, M. R., Wei, S., Posner, B. A., Unger, R. H., Roth, M. G. Tags: Original Research Source Type: research
High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport
The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of T...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Hanson, M., Jordan, L. D., Shipelskiy, Y., Newton, S. M., Klebba, P. E. Tags: Technical Note Source Type: research
Advantages and Challenges of Phenotypic Screens: The Identification of Two Novel Antifungal Geranylgeranyltransferase I Inhibitors
Phenotypic screens are effective starting points to identify compounds with desirable activities. To find novel antifungals, we conducted a phenotypic screen in Saccharomyces cerevisiae and identified two discrete scaffolds with good growth inhibitory characteristics. Lack of broad-spectrum activity against pathogenic fungi called for directed chemical compound optimization requiring knowledge of the molecular target. Chemogenomic profiling identified effects on geranylgeranyltransferase I (GGTase I), an essential enzyme that prenylates proteins involved in cell signaling, such as Cdc42p and Rho1p. Selection of resistant m...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Pries, V., Cotesta, S., Riedl, R., Aust, T., Schuierer, S., Tao, J., Filipuzzi, I., Hoepfner, D. Tags: Original Research Source Type: research
New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories
We describe the development of these assays, focusing on the parameters that were critical for optimization, in the hopes that the lessons learned can be generalized to the development of new biosensor-based assays. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Tewson, P. H., Martinka, S., Shaner, N. C., Hughes, T. E., Quinn, A. M. Tags: Original Research Source Type: research
A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5
We report the development of radioactivity-based assays for kinetic characterization of m6A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z' factors of 0.78 and 0.77, respectively. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Li, F., Kennedy, S., Hajian, T., Gibson, E., Seitova, A., Xu, C., Arrowsmith, C. H., Vedadi, M. Tags: Original Research Source Type: research
Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor
Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Wilson, B. A. P., Alam, M. S., Guszczynski, T., Jakob, M., Shenoy, S. R., Mitchell, C. A., Goncharova, E. I., Evans, J. R., Wipf, P., Liu, G., Ashwell, J. D., OKeefe, B. R. Tags: Original Research Source Type: research
Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions
The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that meas...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Agarinis, C., Orsini, V., Megel, P., Abraham, Y., Yang, H., Mickanin, C., Myer, V., Bouwmeester, T., Tchorz, J. S., Parker, C. N. Tags: Original Research Source Type: research
A High-Content Imaging Screen for Cellular Regulators of {beta}-Catenin Protein Abundance
This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Zeng, X., Montoute, M., Bee, T. W., Lin, H., Kallal, L. A., Liu, Y., Agarwal, P., Wang, D., Lu, Q., Morrow, D., Pope, A. J., Wu, Z. Tags: Original Research Source Type: research
High-Content Analysis of Breast Cancer Using Single-Cell Deep Transfer Learning
High-content analysis has revolutionized cancer drug discovery by identifying substances that alter the phenotype of a cell, which prevents tumor growth and metastasis. The high-resolution biofluorescence images from assays allow precise quantitative measures enabling the distinction of small molecules of a host cell from a tumor. In this work, we are particularly interested in the application of deep neural networks (DNNs), a cutting-edge machine learning method, to the classification of compounds in chemical mechanisms of action (MOAs). Compound classification has been performed using image-based profiling methods someti...
Source: Journal of Biomolecular Screening - February 19, 2016 Category: Molecular Biology Authors: Kandaswamy, C., Silva, L. M., Alexandre, L. A., Santos, J. M. Tags: Original Research Source Type: research
