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Journal of Biomolecular Screening

Andreas Stengl, David Hörl, Heinrich Leonhardt, Jonas Helma
Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats...
December 1, 2016: Journal of Biomolecular Screening
Elisabeth Weber, Ina Rothenaigner, Stefanie Brandner, Kamyar Hadian, Kenji Schorpp
The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer...
December 1, 2016: Journal of Biomolecular Screening
Ioannis K Moutsatsos, Imtiaz Hossain, Claudia Agarinis, Fred Harbinski, Yann Abraham, Luc Dobler, Xian Zhang, Christopher J Wilson, Jeremy L Jenkins, Nicholas Holway, John Tallarico, Christian N Parker
High-throughput screening generates large volumes of heterogeneous data that require a diverse set of computational tools for management, processing, and analysis. Building integrated, scalable, and robust computational workflows for such applications is challenging but highly valuable. Scientific data integration and pipelining facilitate standardized data processing, collaboration, and reuse of best practices. We describe how Jenkins-CI, an "off-the-shelf," open-source, continuous integration system, is used to build pipelines for processing images and associated data from high-content screening (HCS)...
November 29, 2016: Journal of Biomolecular Screening
Tory M Schaaf, Kurt C Peterson, Benjamin D Grant, Prachi Bawaskar, Samantha Yuen, Ji Li, Joseph M Muretta, Gregory D Gillispie, David D Thomas
A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells...
November 29, 2016: Journal of Biomolecular Screening
Tory M Schaaf, Kurt C Peterson, Benjamin D Grant, David D Thomas, Gregory D Gillispie
We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells...
November 22, 2016: Journal of Biomolecular Screening
Won Jae Lee, Dubravka Škalamera, Mareike Dahmer-Heath, Konstanin Shakhbazov, Max V Ranall, Carly Fox, Duncan Lambie, Alexander J Stevenson, Paul Yaswen, Thomas J Gonda, Brian Gabrielli
Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene (HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins...
November 21, 2016: Journal of Biomolecular Screening
Stephanie O Palmer, Yanmei Hu, Megan Keniry, James M Bullard
Four inhibitory compounds were identified using a poly-uridylic acid (polyU) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa in an in vitro screen of a synthetic compound library. The compounds were specific for inhibition of bacterial protein synthesis. In enzymatic assays, the compounds inhibited protein synthesis with IC50 values ranging from 20 to 60 μM. Minimum inhibitory concentrations (MICs) were determined in cultures for a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, P...
November 21, 2016: Journal of Biomolecular Screening
Jing Wang, Pengfei Fang, Peter Chase, Sagi Tshori, Ehud Razin, Timothy P Spicer, Louis Scampavia, Peter Hodder, Min Guo
Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro...
November 8, 2016: Journal of Biomolecular Screening
John R Veloria, Ashwini K Devkota, Eun Jeong Cho, Kevin N Dalby
Apyrase is a calcium-activated enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and Pi It is currently used in studies involving cancer and platelet aggregation in humans, as well as herbicide resistance in plants. Inhibitors of apyrase are being investigated for their use to suppress tumors and combat herbicide resistance. Only a few inhibitors of apyrase have been reported, many of which were identified through automated screening using a 96-well plate format and colorimetric phosphate detection...
November 7, 2016: Journal of Biomolecular Screening
Jinyeong Heo, Jiyoun Nam, Jichan Jang, David Shum, Constantin Radu, Jinhua Cheng, Hanki Lee, Joo-Won Suh, Vincent Delorme
The feasibility and relevance of screening a library of raw actinomycete extracts (ECUM library) for the identification of antituberculosis activities was assessed on 11,088 extracts using a multiple-screening approach. Each extract was first tested at two concentrations against noninfected macrophages as a control, then against Mycobacterium tuberculosis growing in broth medium as well as infecting murine macrophages. The screening results indicated a library of good quality with an apparent low proportion of cytotoxic extracts...
November 3, 2016: Journal of Biomolecular Screening
Donna Leippe, Mary Sobol, Gediminas Vidugiris, James J Cali, Jolanta Vidugiriene
Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis...
November 1, 2016: Journal of Biomolecular Screening
Elizabeth B Rex, Nikhil Shukla, Shenyan Gu, David Bredt, Daniel DiSepio
Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion. Using this approach in combination with a functional assay, we identified positive modulators of α7nAChR activity...
October 27, 2016: Journal of Biomolecular Screening
Göran Dahl, Stephan Steigele, Per Hillertz, Anna Tigerström, Anders Egnéus, Alexander Mehrle, Martin Ginkel, Fredrik Edfeldt, Geoff Holdgate, Nichole O'Connell, Bernd Kappler, Annette Brodte, Philip B Rawlins, Gareth Davies, Eva-Lotta Westberg, Rutger H A Folmer, Stephan Heyse
Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data...
October 27, 2016: Journal of Biomolecular Screening
Srinivasan Krishnan, Mariana C Fiori, Ty E Whisenant, D Marien Cortes, Guillermo A Altenberg, Luis G Cuello
Connexins form the gap junctional channels that mediate cell-to-cell communication, and also form hemichannels present at the plasma membrane. Hemichannels are permeable to small hydrophilic compounds, including molecules involved in autocrine and paracrine signaling. An abnormal hemichannel opening causes or contributes to cell damage in common human disorders (e.g., cardiac infarct, cerebrovascular accidents, deafness, skin diseases, and cataracts) and is therefore a potential pharmacological target. The discovery of useful hemichannels inhibitors has been hampered in part by the lack of suitable high-throughput functional assays...
October 27, 2016: Journal of Biomolecular Screening
Robyn T Rebbeck, Maram M Essawy, Florentin R Nitu, Benjamin D Grant, Gregory D Gillispie, David D Thomas, Donald M Bers, Razvan L Cornea
Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca(2+) channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca(2+) regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca(2+)], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders...
October 19, 2016: Journal of Biomolecular Screening
Markus List, Marlene Pedersen Elnegaard, Steffen Schmidt, Helle Christiansen, Qihua Tan, Jan Mollenhauer, Jan Baumbach
High-throughput screening (HTS) has become an indispensable tool for the pharmaceutical industry and for biomedical research. A high degree of automation allows for experiments in the range of a few hundred up to several hundred thousand to be performed in close succession. The basis for such screens are molecular libraries, that is, microtiter plates with solubilized reagents such as siRNAs, shRNAs, miRNA inhibitors or mimics, and sgRNAs, or small compounds, that is, drugs. These reagents are typically condensed to provide enough material for covering several screens...
October 11, 2016: Journal of Biomolecular Screening
Gyongseon Yang, Nakyung Lee, Jean-Robert Ioset, Joo Hwan No
In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a β-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50 values from two assays showed a very poor correlation, with an R(2) value of 0...
October 11, 2016: Journal of Biomolecular Screening
Patricia Villacé, Rosa M Mella, Meritxell Roura-Ferrer, María Valcárcel, Clarisa Salado, Amaia Castilla, Danel Kortazar
Parkinson disease (PD) is a prevalent neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra, causing tremor and motor impairment. Parkin protein, whose mutants are the cause of Parkinson disease type 2 (PARK2), has been mechanistically linked to the regulation of apoptosis and the turnover of damaged mitochondria. Several studies have implicated aberrant mitochondria as a key contributor to the development of PD. In the attempt to discover new drugs, high-content cell-based assays are becoming more important to mimic the nature of biological processes and their diversifications in diseases and will be essential for lead identification and the optimization of therapeutic candidates...
October 4, 2016: Journal of Biomolecular Screening
Hi Chul Kim, David Shum, Hyang Sook Seol, Se Jin Jang, Ssang-Goo Cho, Yong-Jun Kwon
Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing...
October 4, 2016: Journal of Biomolecular Screening
John M Strelow
The clinical and commercial success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochemical, cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochemical assay, the IC50 may simply reflect the target protein concentration in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI The kinact/KI is a rate constant describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the maximum potential rate (kinact) of inactivation...
October 4, 2016: Journal of Biomolecular Screening
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