scholarly journals A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

2011 ◽  
Vol 16 (8) ◽  
pp. 836-844 ◽  
Author(s):  
Li Li ◽  
Yuhong Du ◽  
Wei Chen ◽  
Haian Fu ◽  
David G. Harrison
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Regulatory Protein ◽  
Resonance Energy ◽  
No Production ◽  
Gtp Cyclohydrolase ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Pharmacologically Active

Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH4 has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH4. The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH4 levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH4 levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z′ factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein–protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH4 levels.

scholarly journals Identification of Small Molecules That Inhibit the Interaction of TEM8 with Anthrax Protective Antigen Using a FRET Assay

2013 ◽  
Vol 18 (6) ◽  
pp. 714-725 ◽  
Author(s):  
Lorna M. Cryan ◽  
Kaiane A. Habeshian ◽  
Thomas P. Caldwell ◽  
Meredith T. Morris ◽  
P. Christine Ackroyd ◽  
...  
Keyword(s):  
Blood Vessels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protective Antigen ◽  
Resonance Energy ◽  
Cysteine Residue ◽  
Tumor Formation ◽  
Screening Assay ◽  
High Throughput Screening Assay

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z’ value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

scholarly journals TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

2011 ◽  
Vol 17 (2) ◽  
pp. 163-176 ◽  
Author(s):  
Charitha Madiraju ◽  
Kate Welsh ◽  
Michael P. Cuddy ◽  
Paulo H. Godoi ◽  
Ian Pass ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Inflammatory Diseases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chemical Library ◽  
Screening Assay ◽  
Time Resolved ◽  
High Throughput Screening Assay

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63–linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)–conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)–conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z′ scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

scholarly journals Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

2003 ◽  
Vol 69 (3) ◽  
pp. 1492-1498 ◽  
Author(s):  
Veit Bergendahl ◽  
Tomasz Heyduk ◽  
Richard R. Burgess
Keyword(s):  
Energy Transfer ◽  
Drug Discovery ◽  
Rna Polymerase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Screening Assay ◽  
Essential Protein ◽  
High Throughput Screening Assay

ABSTRACT The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

scholarly journals A High-Throughput Screening Assay for Fungicidal Compounds against Cryptococcus neoformans

2013 ◽  
Vol 19 (2) ◽  
pp. 270-277 ◽  
Author(s):  
Jennifer L. A. Rabjohns ◽  
Yoon-Dong Park ◽  
Jean Dehdashti ◽  
Wei Sun ◽  
Christina Henderson ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
High Throughput ◽  
Cryptococcal Meningitis ◽  
High Throughput Screening ◽  
Animal Studies ◽  
Pathogenic Fungus ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)–infected individuals. Although amphotericin B is the “gold standard” treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.

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