The King Is Dead, Long Live the King! JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Bickle, M., Djaballah, H., Mayr, L. M. Tags: Articles Source Type: research
Corrigendum
R. Jeffrey Neitz, Steven Chen, Frantisek Supek, Vince Yeh, Danielle Kellar, Jiri Gut, Clifford Bryant, Alejandra Gallardo-Godoy, Valentina Molteni, Steven L. Roach, Arnab K. Chatterjee, Stephanie Robertson, Adam R. Renslo, Michelle Arkin, Richard Glynne, James McKerrow, and Jair L. Siqueira-Neto. Lead Identification to Clinical Candidate Selection: Drugs for Chagas Disease J. Biomol. Screen. 2015, 20, 101–111. (Original doi:10.1177/1087057114553103)
In the January 2015 issue of the Journal of Biomolecular Screening, the names of two authors were not published with this article, Shilpi Khare and Monique Stinson. Their...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Tags: Corrigendum Source Type: research
The Bacterial Sec Pathway of Protein Export: Screening and Follow-Up
Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers’ focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (β-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Crowther, G. J., Weller, S. M., Jones, J. C., Weaver, T., Fan, E., Van Voorhis, W. C., Rosen, H. Tags: Technical Notes Source Type: research
Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors
A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular ...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Rosenberg, L. H., Lafitte, M., Grant, W., Chen, W., Cleveland, J. L., Duckett, D. R. Tags: Technical Notes Source Type: research
Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay
In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Xu, Y., Wang, Y., Xu, Y., Li, J., Liao, H., Zhang, L., Pang, T. Tags: Technical Notes Source Type: research
Meltdown: A Tool to Help in the Interpretation of Thermal Melt Curves Acquired by Differential Scanning Fluorimetry
The output of a differential scanning fluorimetry (DSF) assay is a series of melt curves, which need to be interpreted to get value from the assay. An application that translates raw thermal melt curve data into more easily assimilated knowledge is described. This program, called "Meltdown," conducts four main activities—control checks, curve normalization, outlier rejection, and melt temperature (Tm) estimation—and performs optimally in the presence of triplicate (or higher) sample data. The final output is a report that summarizes the results of a DSF experiment. The goal of Meltdown is not to replace human a...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Rosa, N., Ristic, M., Seabrook, S. A., Lovell, D., Lucent, D., Newman, J. Tags: Application Note Source Type: research
A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays
A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise–filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Hsieh, J.-H., Sedykh, A., Huang, R., Xia, M., Tice, R. R. Tags: Original Research Source Type: research
Phenotypic Approaches to Identify Inhibitors of B Cell Activation
An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin’s lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton’s tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compar...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Rex, E. B., Kim, S., Wiener, J., Rao, N. L., Milla, M. E., DiSepio, D. Tags: Original Research Source Type: research
A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening
For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody–drug conjugates. Here we describe a novel activatable fluorescence–qu...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Li, Y., Liu, P. C., Shen, Y., Snavely, M. D., Hiraga, K. Tags: Original Research Source Type: research
Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4)
Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer’s disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M4 (M4), we developed a homogenous, multiparametric, 1536-well assay to measure M4 receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z' of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Smith, E., Chase, P., Niswender, C. M., Utley, T. J., Sheffler, D. J., Noetzel, M. J., Lamsal, A., Wood, M. R., Conn, P. J., Lindsley, C. W., Madoux, F., Acosta, M., Scampavia, L., Spicer, T., Hodder, P. Tags: Original Research Source Type: research
A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors
Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein–coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)–based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in livin...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Vedel, L., Brauner-Osborne, H., Mathiesen, J. M. Tags: Original Research Source Type: research
Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry
This article describes the use of self-assembled monolayer desorption/ionization mass spectrometry, or SAMDI-MS—a label-free drug discovery tool—to characterize SIRT3 activity and discover inhibitors. SAMDI-MS was used to analyze a peptide array having 361 distinct acetylated peptides to identify an active SIRT3 substrate (GYKAcRGC). This peptide was used in a screen of 100,000 small molecules to identify inhibitors of SIRT3. A total of 306 SIRT3 inhibitors were identified, with one compound, SDX-437, having an IC50 of 700 nM with >100-fold selectivity for SIRT3 over SIRT1. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Patel, K., Sherrill, J., Mrksich, M., Scholle, M. D. Tags: Original Research Source Type: research
DNA Repair Endonucleases: Physiological Roles and Potential as Drug Targets
Genomic DNA is constantly exposed to endogenous and exogenous damaging agents. To overcome these damaging effects and maintain genomic stability, cells have robust coping mechanisms in place, including repair of the damaged DNA. There are a number of DNA repair pathways available to cells dependent on the type of damage induced. The removal of damaged DNA is essential to allow successful repair. Removal of DNA strands is achieved by nucleases. Exonucleases are those that progressively cut from DNA ends, and endonucleases make single incisions within strands of DNA. This review focuses on the group of endonucleases involved...
Source: Journal of Biomolecular Screening - July 20, 2015 Category: Molecular Biology Authors: Doherty, R., Madhusudan, S. Tags: Review Article Source Type: research
Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening
Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)–based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 compounds in the HitFinder collection v.11 were screened, which represent all the distinct skeletons of the Maybridge Librar...
Source: Journal of Biomolecular Screening - June 19, 2015 Category: Molecular Biology Authors: Wang, W., Marholz, L. J., Wang, X. Tags: Technical Note Source Type: research
A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening
In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify c...
Source: Journal of Biomolecular Screening - June 19, 2015 Category: Molecular Biology Authors: Wigle, T. J., Swinger, K. K., Campbell, J. E., Scholle, M. D., Sherrill, J., Admirand, E. A., Boriack-Sjodin, P. A., Kuntz, K. W., Chesworth, R., Moyer, M. P., Scott, M. P., Copeland, R. A. Tags: Original Research Source Type: research
