Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists
G protein–coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phospha...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Zhu, Y., Watson, J., Chen, M., Shen, D. R., Yarde, M., Agler, M., Burford, N., Alt, A., Jayachandra, S., Cvijic, M. E., Zhang, L., Dyckman, A., Xie, J., O'Connell, J., Banks, M., Weston, A. Tags: Original Research Source Type: research
Development of a Kinetic Assay for Late Endosome Movement
Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells ...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Esner, M., Meyenhofer, F., Kuhn, M., Thomas, M., Kalaidzidis, Y., Bickle, M. Tags: Original Research Source Type: research
Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2
Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)–based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Wyhs, N., Walker, D., Giovinazzo, H., Yegnasubramanian, S., Nelson, W. G. Tags: Original Research Source Type: research
The Resazurin Reduction Assay Can Distinguish Cytotoxic from Cytostatic Compounds in Spheroid Screening Assays
In conclusion, the RR assay is unsuited to quantitatively measure cellular health/cell number in compact spheroids. However, it can be used to distinguish between cytotoxic versus cytostatic compounds in spheroids. Restoration of the correlation of cell viability/number to resazurin reduction capacity can be achieved by disruption of tight junctions. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Walzl, A., Unger, C., Kramer, N., Unterleuthner, D., Scherzer, M., Hengstschlager, M., Schwanzer-Pfeiffer, D., Dolznig, H. Tags: Original Research Source Type: research
Identification of Small Inhibitory Molecules Targeting the Bfl-1 Anti-Apoptotic Protein That Alleviates Resistance to ABT-737
One approach currently being developed in anticancer drug discovery is to search for small compounds capable of occupying and blocking the hydrophobic pocket of anti-apoptotic Bcl-2 family members necessary for interacting with pro-apoptotic proteins. Such an approach led to the discovery of several compounds, such as ABT-737 (which interacts with Bcl-2, Bcl-xl, and Bcl-w) or the latest one, ABT-199, that selectively targets Bcl-2 protein. The efficacy of those compounds is, however, limited by the expression of two other anti-apoptotic Bcl-2 members, Mcl-1 and Bfl-1. Based on the role of Bfl-1 in cancer, especially in che...
Source: Journal of Biomolecular Screening - July 18, 2014 Category: Molecular Biology Authors: Mathieu, A.-L., Sperandio, O., Pottiez, V., Balzarin, S., Herledan, A., Elkaim, J. O., Fogeron, M.-L., Piveteau, C., Dassonneville, S., Deprez, B., Villoutreix, B. O., Bonnefoy, N., Leroux, F. Tags: Original Research Source Type: research
