A Platform to Enable the Pharmacological Profiling of Small Molecules in Gel-Based Electrophoretic Mobility Shift Assays
We describe a polyacrylamide gel casting cassette that overcomes limitations of commercially available gel electrophoresis equipment. This apparatus molds a single polyacrylamide gel that can evaluate more than 200 samples in parallel, is loaded with a multichannel pipettor, and is flexible with respect to composition of the separating matrix. We demonstrate its use to characterize inhibitors of enzymes that modify protein and nucleic acid substrates. Throughputs of greater than 1000 samples per day were achieved when this system was paired with a quantitative laser-based imaging system, yielding data of remarkable quality...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Foley, T. L., Dorjsuren, D., Dexheimer, T. S., Burkart, M. D., Wight, W. C., Simeonov, A. Tags: Technical Note Source Type: research
One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays
Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate precoating, hampering its applications in high-throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human ...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Dai, S., Li, R., Long, Y., Titus, S., Zhao, J., Huang, R., Xia, M., Zheng, W. Tags: Original Research Source Type: research
Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein-Protein Interactions
Protein interaction networks are the basis for human metabolic and signaling systems. Interaction studies often use bimolecular fluorescence complementation (BiFC) to reveal the formation and cellular localization of protein complexes. However, large-scale studies were either far from native conditions in human cells or limited by laborious restriction/ligation cloning techniques. Here, we describe a new tool for protein interaction screening based on Gateway-compatible BiFC vectors. We made a set of four new vectors that permit fusion of candidate proteins to the N or C fragment of Venus in all fusion positions. We have v...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Lepur, A., Kovacevic, L., Beluzic, R., Vugrek, O. Tags: Original Research Source Type: research
Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs
Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone–related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have co...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Wang, Z., Chen, Y., Drbohlav, L. M., Wu, J. Q., Wang, M. Z. Tags: Original Research Source Type: research
Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived from High-Throughput Screening
One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to ...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Auld, D. S., Jimenez, M., Yue, K., Busby, S., Chen, Y.-C., Bowes, S., Wendel, G., Smith, T., Zhang, J.-H. Tags: Original Research Source Type: research
Functional Characterization of Acetylcholine Receptors Expressed in Human Neurons Differentiated from Hippocampal Neural Stem/Progenitor Cells
Neurotransmission mediated by acetylcholine receptors (AChRs) plays an important role in learning and memory functions in the hippocampus. Impairment of the cholinergic system contributes to Alzheimer’s disease (AD), indicating the importance of AChRs as drug targets for AD. To improve the success rates for AD drug development, human cell models that mimic the target brain region are important. Therefore, we characterized the functional expression of nicotinic and muscarinic AChRs (nAChRs and mAChRs, respectively) in human hippocampal neurons differentiated from hippocampal neural stem/progenitor cells (HIP-009 cells...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Fukushima, K., Yamazaki, K., Miyamoto, N., Sawada, K. Tags: Original Research Source Type: research
Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions
Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neuron...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Yamazaki, K., Fukushima, K., Sugawara, M., Tabata, Y., Imaizumi, Y., Ishihara, Y., Ito, M., Tsukahara, K., Kohyama, J., Okano, H. Tags: Original Research Source Type: research
Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay
Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent ...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Stead, C., Brown, A., Adams, C., Nickolls, S. J., Young, G., Kammonen, J., Pryde, D., Cawkill, D. Tags: Original Research Source Type: research
In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors
PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escheric...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Salowe, S. P., Zhang, L., Zokian, H. J., Gesell, J. J., Zink, D. L., Wiltsie, J., Ai, X., Kavana, M., Pinto, S. Tags: Original Research Source Type: research
Early Perspective: Microplate Applications of the Cellular Thermal Shift Assay (CETSA)
The cellular thermal shift assay (CETSA) was introduced in 2013 as a means to assess drug binding in complex environments such as cell lysates, live cells, and even tissues. The assay principle relies on the well-proven biophysical concept of ligand-induced thermal stabilization of proteins, which in CETSA applications is measured as a persistent presence of soluble protein at elevated temperatures. Given its recent development, we have just started to learn about the benefits and pitfalls of the method as it is applied to a growing number of protein target classes, the majority of which are intracellular soluble proteins....
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Seashore-Ludlow, B., Lundbäck, T. Tags: Perspective Article Source Type: research
Rethinking Nuclear Receptors as Potential Therapeutic Targets for Retinal Diseases
Collectively, retinal diseases, including age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy, result in severe vision impairment worldwide. The absence and/or limited availability of successful drug therapies for these blinding disorders necessitates further understanding their pathobiology and identifying new targetable signaling pathways. Nuclear receptors are transcription regulators of many key aspects of human physiology, as well as pathophysiology, with reported roles in development, aging, and disease. Some of the pathways regulated by nuclear receptors include, but are not limited to, ...
Source: Journal of Biomolecular Screening - November 8, 2016 Category: Molecular Biology Authors: Choudhary, M., Malek, G. Tags: Review Source Type: research
Single-Cell Phenotype Classification Using Deep Convolutional Neural Networks
Deep learning methods are currently outperforming traditional state-of-the-art computer vision algorithms in diverse applications and recently even surpassed human performance in object recognition. Here we demonstrate the potential of deep learning methods to high-content screening–based phenotype classification. We trained a deep learning classifier in the form of convolutional neural networks with approximately 40,000 publicly available single-cell images from samples treated with compounds from four classes known to lead to different phenotypes. The input data consisted of multichannel images. The construction of...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Dürr, O., Sick, B. Tags: Technical Notes Source Type: research
Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context
The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression–based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) a...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Severyn, B., Nguyen, T., Altman, M. D., Li, L., Nagashima, K., Naumov, G. N., Sathyanarayanan, S., Cook, E., Morris, E., Ferrer, M., Arthur, B., Benita, Y., Watters, J., Loboda, A., Hermes, J., Gilliland, D. G., Cleary, M. A., Carroll, P. M., Strack, P., Tags: Technical Notes Source Type: research
Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening
Production of type I interferon (IFN) is an essential component of the innate immune response against invading pathogens. However, its production must be tightly regulated to avoid harmful effects. Compounds that modulate the IFN response are potentially valuable for a variety of applications due to IFN’s beneficial and detrimental roles. We developed and executed a cell-based high-throughput screen (HTS) targeting components that participate in and/or regulate the IRF3 and nuclear factor (NF)–B branches of the IFN induction pathway. The assay detects activation of the IFN induction pathway via an enhanced gree...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Gage, Z. O., Vasou, A., Gray, D. W., Randall, R. E., Adamson, C. S. Tags: Original Research Source Type: research
A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells
This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Schorpp, K., Rothenaigner, I., Maier, J., Traenkle, B., Rothbauer, U., Hadian, K. Tags: Original Research Source Type: research
