Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format
Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer’s disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproduci...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Medda, X., Mertens, L., Versweyveld, S., Diels, A., Barnham, L., Bretteville, A., Buist, A., Verheyen, A., Royaux, I., Ebneth, A., Cabrera-Socorro, A. Tags: Original Research Source Type: research
Morphological Evaluation of Nonlabeled Cells to Detect Stimulation of Nerve Growth Factor Expression by Lyconadin B
In this study, we applied morphology-based evaluation of nonlabeled microscopic images to a phenotype-based assay. As a study case, we attempted to increase the efficiency of a cell-based assay for chemical compounds that induce production of nerve growth factor (NGF), using lyconadin B as a model compound. Because the total synthesis of lyconadin B was accomplished very recently, there is no well-established cell-based assay scheme for further drug screening. The conventional cell-based assay for evaluating NGF induction requires two types of cells and a total of 5 days of cell culture. The complexity and length of this a...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Kawai, S., Sasaki, H., Okada, N., Kanie, K., Yokoshima, S., Fukuyama, T., Honda, H., Kato, R. Tags: Original Research Source Type: research
The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8
In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Strelow, J. M., Xiao, M., Cavitt, R. N., Fite, N. C., Margolis, B. J., Park, K.-J. Tags: Original Research Source Type: research
Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer Therapies
Evidence of adaptive immune responses in the prevention of cancer has been accumulating for decades. Spontaneous T-cell responses occur in multiple indications, bringing the study of de novo expressed cancer antigens to the fore and highlighting their potential as targets for cancer immunotherapy. Circumventing the immune-suppressive mechanisms that maintain tumor tolerance and driving an antitumor cytotoxic T-cell response in cancer patients may eradicate the tumor or block disease progression. Multiple strategies are being pursued to harness the cytotoxic potential of T cells clinically. Highly promising results are now ...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Hickman, E. S., Lomax, M. E., Jakobsen, B. K. Tags: Review Source Type: research
Erratum
The guest editors of the June 2016 special issue of the Journal of Biomolecular Screening (Vol. 21, No. 5) (JBS) on "Innovative Screening Methodologies to Identify New Compounds for the Treatment of Central Nervous System Disorders" were not acknowledged in the final published issue. JBS gratefully thanks Drs. Kevin Burris and Steven Dworetzky for their valued contributions and apologizes for the oversight. (Original DOI: 10.1177/1087057116644231). Please visit the table of contents for this special issue at http://jbx.sagepub.com/content/21/5.toc. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Tags: Other Source Type: research
Screening for Non-Pore-Binding Modulators of EAG K+ Channels
In this report, we present two independent screening campaigns in which we wanted to identify small molecules that bind to either the intracellular cytoplasmic amino terminal Per-Arnt-Sim (PAS) domain from the human EAG-related gene (ERG) channel or the amino or carboxy terminal globular domains from the mouse EAG1 channel, affecting their interaction. We report that in both cases, compounds were identified that showed weak, nonspecific binding. We suggest alternative routes should be pursued in future efforts to identify specific, high-affinity binders to these cytoplasmic domains. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Fernandes, A. S., Morais-Cabral, J. H., Harley, C. A. Tags: Technical Note Source Type: research
A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors
Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Stravalaci, M., De Blasio, D., Orsini, F., Perego, C., Palmioli, A., Goti, G., Bernardi, A., De Simoni, M.-G., Gobbi, M. Tags: Original Research Source Type: research
Development of a High-Throughput Screening Strategy for Upregulators of the OPG/RANKL Ratio with the Potential for Antiosteoporosis Effects
In this study, we established a novel high-throughput screening (HTS) system using two stably transfected monoclonal cell lines that either express firefly luciferase under the OPG promoter control or concurrently express firefly and renilla luciferases under control of the OPG and RANKL promoters, respectively. With this system, we can conveniently and rapidly detect the effects of compounds on the expression of OPG and RANKL through changes in firefly and renilla luciferase activities. A total of 8160 compounds were screened using this system, yielding five compounds without previously reported activity. The compound wit...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Gong, S., Han, X., Li, X., Yang, J., He, X., Si, S. Tags: Original Research Source Type: research
Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach
The dissociation rates of unlabeled drugs have been well studied by kinetic binding analyses. Since kinetic assays are laborious, we developed a simple method to determine the kinetic binding parameters of unlabeled competitors by a preincubation endpoint assay. The probe binding after preincubation of a competitor can be described by a single equation as a function of time. Simulations using the equation revealed the degree of IC50 change induced by preincubation of a competitor depended on the dissociation rate koff of the competitor but not on the association rate kon. To validate the model, an in vitro binding assay wa...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Shimizu, Y., Ogawa, K., Nakayama, M. Tags: Original Research Source Type: research
A Quantitative Spectrophotometric Assay to Monitor the tRNA-Dependent Pathway for Lipid Aminoacylation In Vitro
The transfer RNA (tRNA)–dependent pathway for lipid aminoacylation is a two-step pathway composed of (1) a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase, forming a specific aa-tRNA, and (2) a tRNA-dependent transfer step in which the amino acid acylating the tRNA is transferred to an acceptor lipid. The latter step is catalyzed by a transferase located within the cytoplasmic membrane of certain bacteria. Lipid aminoacylation modifies the biochemical properties of the membrane and enhances resistance of some pathogens to various classes of antimicrobial agents and components of the innate immune r...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Grube, C. D., Roy, H. Tags: Original Research Source Type: research
Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles
This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Ewing, H., Fernandez-Vega, V., Spicer, T. P., Chase, P., Brown, S., Scampavia, L., Roush, W. R., Riley, S., Rosen, H., Hodder, P., Lambeau, G., Gelb, M. H. Tags: Original Research Source Type: research
FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay: New Internally Quenched Fluorogenic Substrates for High-Throughput Screening
In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Caporale, A., Mascanzoni, F., Farina, B., Sturlese, M., Di Sorbo, G., Fattorusso, R., Ruvo, M., Doti, N. Tags: Original Research Source Type: research
Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target
We report the development and implementation of a high-throughput luciferase-based activity assay and screening of 19,920 compounds derived from small-molecule libraries and an in silico screen predicting likely inhibitors of the cytidylate kinase enzyme. Hit validation included a counterscreen for luciferase inhibitors that would result in false positives in the initial screen. Results of this counterscreen ruled out all of the putative cytidylate kinase inhibitors identified in the initial screening, leaving no compounds as candidates for drug development. Although a negative result, this study indicates that this import...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Craig, J. K., Risler, J. K., Loesch, K. A., Dong, W., Baker, D., Barrett, L. K., Subramanian, S., Samudrala, R., Van Voorhis, W. C. Tags: Original Research Source Type: research
Bacterial Expression and HTS Assessment of Soluble Epoxide Hydrolase Phosphatase
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were ident...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Klingler, F.-M., Wolf, M., Wittmann, S., Gribbon, P., Proschak, E. Tags: Original Research Source Type: research
Transcriptional Inhibitors Identified in a 160,000-Compound Small-Molecule DUX4 Viability Screen
Facioscapulohumeral muscular dystrophy is a genetically dominant, currently untreatable muscular dystrophy. It is caused by mutations that enable expression of the normally silent DUX4 gene, which encodes a pathogenic transcription factor. A screen based on Tet-on DUX4-induced mouse myoblast death previously uncovered compounds from a 44,000-compound library that protect against DUX4 toxicity. Many of those compounds acted downstream of DUX4 in an oxidative stress pathway. Here, we extend this screen to an additional 160,000 compounds and, using greater stringency, identify a new set of DUX4-protective compounds. From 640 ...
Source: Journal of Biomolecular Screening - July 19, 2016 Category: Molecular Biology Authors: Choi, S. H., Bosnakovski, D., Strasser, J. M., Toso, E. A., Walters, M. A., Kyba, M. Tags: Original Research Source Type: research
