Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments
The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the bindi...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Della Ducata, D., Jaehrling, J., Hänel, C., Satzger, M., Wolber, M., Ostendorp, R., Pabst, S., Brocks, B. Tags: Original Research Source Type: research
Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants
A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3–mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small lib...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Reinen, J., van Hemert, D., Vermeulen, N. P. E., Commandeur, J. N. M. Tags: Original Research Source Type: research
Cell-Based High-Throughput Luciferase Reporter Gene Assays for Identifying and Profiling Chemical Modulators of Endoplasmic Reticulum Signaling Protein, IRE1
Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the "adaptive" signaling cascade mediated by the XBP1 pathway and the "alarm" signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HT...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Rong, J., Pass, I., Diaz, P. W., Ngo, T. A., Sauer, M., Magnuson, G., Zeng, F.-Y., Hassig, C. A., Jackson, M. R., Cosford, N. D. P., Matsuzawa, S.-i., Reed, J. C. Tags: Original Research Source Type: research
High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor
Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Terry-Lorenzo, R. T., Masuda, K., Sugao, K., Fang, Q. K., Orsini, M. A., Sacchi, S., Pollegioni, L. Tags: Original Research Source Type: research
Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening
There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mu...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Molinski, S. V., Ahmadi, S., Hung, M., Bear, C. E. Tags: Original Research Source Type: research
Drug Discovery of Therapies for Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is a genetic, lethal, muscle disorder caused by the loss of the muscle protein, dystrophin, leading to progressive loss of muscle fibers and muscle weakness. Drug discovery efforts targeting DMD have used two main approaches: (1) the restoration of dystrophin expression or the expression of a compensatory protein, and (2) the mitigation of downstream pathological mechanisms, including dysregulated calcium homeostasis, oxidative stress, inflammation, fibrosis, and muscle ischemia. The aim of this review is to introduce the disease, its pathophysiology, and the available research tools to a ...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Blat, Y., Blat, S. Tags: Review Article Source Type: research
Automatic 3D Cell Analysis in High-Throughput Microarray Using Micropillar and Microwell Chips
In this study, the optimal way to analyze 3D cultured cells is achieved by comparing day-to-day data of doubling times and IC50 values obtained from the two methods. In experiments, the U251 cell line is grown in chips. The doubling time, based on the area of the 3D cells, was 27.8 ± 1.8 h (standard deviation: 6.6%) and 27.8 ± 3.8 h (standard deviation: 13.7%) based on the intensity of the 3D cells. The doubling time calculated by area shows a smaller standard deviation than one calculated by intensity. IC50 values calculated by both methods are very similar. The standard deviations of IC50 values for the two...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Lee, D. W., Lee, M.-Y., Ku, B., Nam, D.-H. Tags: Technical Notes Source Type: research
Development and Application of a High-Throughput Screening Method to Evaluate Antifungal Activity against Trichophyton tonsurans
There exist relatively few drug classes on the market to treat dermatophyte infections. This investigation was designed to develop and validate high-throughput methodology for screening and confirmation of chemicals for activity against Trichophyton tonsurans. Growth characteristics were examined on two platforms (96- and 384-well) in three media at eight spore concentrations over a period of up to 120 h. Microspectrophotometry was used to automate plate reads. The 384-well platform was used to screen more than 7000 compounds from six chemical libraries. Z-scores for optical density relative to positive growth controls wer...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Preuett, B., Leeder, J. S., Abdel-Rahman, S. Tags: Technical Notes Source Type: research
Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa
Pseudomonas aeruginosa glutamyl-tRNA synthetase (GluRS) was overexpressed in Escherichia coli. Sequence analysis indicated that P. aeruginosa GluRS is a discriminating GluRS and, similar to other GluRS proteins, requires the presence of tRNAGlu to produce a glutamyl-AMP intermediate. Kinetic parameters for interaction with tRNA were determined and the kcat and KM were 0.8 s–1 and 0.68 µM, respectively, resulting in a kcat/KM of 1.18 s–1 µM–1. A robust aminoacylation-based scintillation proximity assay (SPA) assay was developed and 800 natural products and 890 synthetic compounds were screened ...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Hu, Y., Guerrero, E., Keniry, M., Manrrique, J., Bullard, J. M. Tags: Original Research Source Type: research
A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation
Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myel...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Radomska, H. S., Jernigan, F., Nakayama, S., Jorge, S. E., Sun, L., Tenen, D. G., Kobayashi, S. S. Tags: Original Research Source Type: research
High-Throughput Screening for Positive Allosteric Modulators Identified Potential Therapeutics against Acetylcholinesterase Inhibition
The current standard of care for treatment of organophosphate (OP) poisoning includes pretreatment with the weak reversible acetylcholinesterase (AChE) inhibitor pyridostigmine bromide. Because this drug is an AChE inhibitor, similar side effects exist as with OP poisoning. In an attempt to provide a therapeutic capable of mitigating AChE inhibition without such side effects, high-throughput screening was performed to identify a compound capable of increasing the catalytic activity of AChE. Herein, two such novel positive allosteric modulators (PAMs) of AChE are presented. These PAMs increase AChE activity threefold, but t...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Chapleau, R. R., McElroy, C. A., Ruark, C. D., Fleming, E. J., Ghering, A. B., Schlager, J. J., Poeppelman, L. D., Gearhart, J. M. Tags: Original Research Source Type: research
Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen
In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense (BDNF-AS) and Hox transcript antisense RNA (HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Pedram Fatemi, R., Salah-Uddin, S., Modarresi, F., Khoury, N., Wahlestedt, C., Faghihi, M. A. Tags: Original Research Source Type: research
Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat–containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain withou...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Han, Y., Lyman, K., Clutter, M., Schiltz, G. E., Ismail, Q.-A., Prados, D. B., Luan, C.-H., Chetkovich, D. M. Tags: Original Research Source Type: research
Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers
According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl– channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Shalaly, N. D., Aneiros, E., Blank, M., Mueller, J., Nyman, E., Blind, M., Dabrowski, M. A., Andersson, C. V., Sandberg, K. Tags: Original Research Source Type: research
High-Throughput Screening Using iPSC-Derived Neuronal Progenitors to Identify Compounds Counteracting Epigenetic Gene Silencing in Fragile X Syndrome
We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Kaufmann, M., Schuffenhauer, A., Fruh, I., Klein, J., Thiemeyer, A., Rigo, P., Gomez-Mancilla, B., Heidinger-Millot, V., Bouwmeester, T., Schopfer, U., Mueller, M., Fodor, B. D., Cobos-Correa, A. Tags: Original Research Source Type: research
