ERK and {beta}-Arrestin Interaction: A Converging Point of Signaling Pathways for Multiple Types of Cell Surface Receptors
β-Arrestin, a signal adaptor protein, mediates intracellular signal transductions through protein-protein interactions by bringing two or more proteins in proximity. Extracellular signal-regulated kinase (ERK), a protein kinase in the family of mitogen-activated protein kinases (MAPKs), is involved in various receptor signal pathways. Interaction of ERK with β-arrestin or formation of ERK/β-arrestin signal complex occurs in response to activation of a variety of cell surface receptors. The ERK/β-arrestin signal complex may be a common transducer to converge a variety of extracellular stimuli to similar ...
Source: Journal of Biomolecular Screening - February 19, 2015 Category: Molecular Biology Authors: Eishingdrelo, H., Sun, W., Li, H., Wang, L., Eishingdrelo, A., Dai, S., McKew, J. C., Zheng, W. Tags: Original Research Source Type: research
A New System for Profiling Drug-Induced Calcium Signal Perturbation in Human Embryonic Stem Cell-Derived Cardiomyocytes
The emergence of human stem cell–derived cardiomyocyte (hSCCM)–based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametr...
Source: Journal of Biomolecular Screening - February 19, 2015 Category: Molecular Biology Authors: Lewis, K. J., Silvester, N. C., Barberini-Jammaers, S., Mason, S. A., Marsh, S. A., Lipka, M., George, C. H. Tags: Original Research Source Type: research
Emergence of Chinese Drug Discovery Research: Impact of Hit and Lead Identification
The identification of hits and the generation of viable leads is an early and yet crucial step in drug discovery. In the West, the main players of drug discovery are pharmaceutical and biotechnology companies, while in China, academic institutions remain central in the field of drug discovery. There has been a tremendous amount of investment from the public as well as private sectors to support infrastructure buildup and expertise consolidation relative to drug discovery and development in the past two decades. A large-scale compound library has been established in China, and a series of high-impact discoveries of lead com...
Source: Journal of Biomolecular Screening - February 19, 2015 Category: Molecular Biology Authors: Zhou, C., Zhou, Y., Wang, J., Zhu, Y., Deng, J., Wang, M.-W. Tags: Review Articles Source Type: research
Addressing the Right Targets in Oncology: Challenges and Alternative Approaches
Translating existing and emerging knowledge of cancer biology into effective novel therapies remains a great challenge in drug discovery. A firm understanding of the target biology, confidence in the supporting preclinical research, and access to diverse chemical matter is required to lower attrition rates and prosecute targets effectively. Understanding past successes and failures will aid in refining this process to deliver further therapeutic benefit to patients. In this review, we suggest that early oncology drug discovery should focus on selection and prosecution of cancer targets with strong disease biology rather th...
Source: Journal of Biomolecular Screening - February 19, 2015 Category: Molecular Biology Authors: Stock, J. K., Jones, N. P., Hammonds, T., Roffey, J., Dillon, C. Tags: Review Articles Source Type: research
Screening Robotics and Automation
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Mattheakis, L. Tags: Product Focus Source Type: research
A Magnetic Bead-Based Ligand Binding Assay to Facilitate Human Kynurenine 3-Monooxygenase Drug Discovery
Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead–based binding assay using mass spectrometr...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Wilson, K., Mole, D. J., Homer, N. Z. M., Iredale, J. P., Auer, M., Webster, S. P. Tags: Technical Notes Source Type: research
Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers
Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are con...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Stylianou, M., Uvell, H., Lopes, J. P., Enquist, P.-A., Elofsson, M., Urban, C. F. Tags: Technical Notes Source Type: research
Streptomyces: A Screening Tool for Bacterial Cell Division Inhibitors
Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the lack of essentiality, the streptomycetes divisome is similar to that of other prokaryotes. Therefore, the chemical inhibitors of sporulation in model streptomycetes may interfere with the cell division in rod-shaped bacteria as well. To test this, we investigated 196 compounds that...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Jani, C., Tocheva, E. I., McAuley, S., Craney, A., Jensen, G. J., Nodwell, J. Tags: Original Research Source Type: research
Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase
Mycobacterium tuberculosis (Mtb) DNA gyrase ATPase was the target of a tuberculosis drug discovery program. The low specific activity of the Mtb ATPase prompted the use of Mycobacterium smegmatis (Msm) enzyme as a surrogate for lead generation, since it had 20-fold higher activity. Addition of GyrA or DNA did not significantly increase the activity of the Msm GyrB ATPase, and an assay was developed using GyrB alone. Inhibition of the Msm ATPase correlated well with inhibition of Mtb DNA gyrase supercoiling across three chemical scaffolds, justifying its use. As the IC50 of compounds approached the enzyme concentration, sur...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Humnabadkar, V., Madhavapeddi, P., Basavarajappa, H., Sheikh, M. G., Rane, R., Basu, R., Verma, P., Sundaram, A., Mukherjee, K., de Sousa, S. M. Tags: Original Research Source Type: research
A Novel In Vitro Approach for Simultaneous Evaluation of CYP3A4 Inhibition and Kinetic Aqueous Solubility
In the early stages of the drug discovery process, evaluation of the drug metabolism and physicochemical properties of new chemical entities is crucial to prioritize those candidates displaying a better profile for further development. In terms of metabolism, drug–drug interactions mediated through CYP450 inhibition are a significant safety concern, and therefore the effect of new candidate drugs on CYP450 activity should be screened early. In the initial stages of drug discovery, when physicochemical properties such as aqueous solubility have not been optimized yet, there might be a large number of candidate compoun...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Perez, J., Diaz, C., Asensio, F., Palafox, A., Genilloud, O., Vicente, F. Tags: Original Research Source Type: research
Validation and Optimization of Novel High-Throughput Assays for Human Epithelial Sodium Channels
The epithelial sodium channel (ENaC) plays a crucial role in salt and water homeostasis and is primarily involved in sodium reabsorption in the kidney and lung. Modulators of ENaC function, particularly within lung epithelia, could offer potential treatments for a number of diseases. As a constitutively active sodium channel, ENaC expression at the cell membrane is highly regulated through rapid turnover. This short half-life of the channel at the membrane and cytotoxicity from overexpression pose a problem for reagent generation and assay development in drug discovery. We have generated an HEK293 stable cell line expressi...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Chen, M. X., Gatfield, K., Ward, E., Downie, D., Sneddon, H. F., Walsh, S., Powell, A. J., Laine, D., Carr, M., Trezise, D. Tags: Original Research Source Type: research
Improving Detection of Rare Biological Events in High-Throughput Screens
We report comparisons among normalization methods in two titration series experiments. We also extend the results in a third experiment with two differently designed but otherwise identical screens: compounds in replicate plates were either placed in the same well locations or were randomly assigned to different locations. Best results were obtained when randomization was combined with normalization methods that corrected for within-plate spatial bias. We conclude that potent, reliable, and accurate HTS requires replication, randomization design strategies, and more extensive normalization than is typically done and that f...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Murie, C., Barette, C., Button, J., Lafanechere, L., Nadon, R. Tags: Original Research Source Type: research
Progesterone Receptor Chaperone Complex-Based High-Throughput Screening Assay: Identification of Capsaicin as an Inhibitor of the Hsp90 Machine
We report here the development of a novel high-throughput screening assay platform to identify small-molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor, a physiological client of Hsp90, in a 96-well plate format. We screened the National Institutes of Health clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is a Food and Drug Administration–approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cel...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Patwardhan, C. A., Alfa, E., Lu, S., Chadli, A. Tags: Original Research Source Type: research
Use of High-Throughput Mass Spectrometry to Reduce False Positives in Protease uHTS Screens
As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)–MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5–7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mas...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Adam, G. C., Meng, J., Rizzo, J. M., Amoss, A., Lusen, J. W., Patel, A., Riley, D., Hunt, R., Zuck, P., Johnson, E. N., Uebele, V. N., Hermes, J. D. Tags: Original Research Source Type: research
Novel High-Throughput Deoxyribonuclease 1 Assay
Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other ...
Source: Journal of Biomolecular Screening - January 22, 2015 Category: Molecular Biology Authors: Jang, D. S., Penthala, N. R., Apostolov, E. O., Wang, X., Fahmi, T., Crooks, P. A., Basnakian, A. G. Tags: Original Research Source Type: research
