scholarly journals Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

2012 ◽  
Vol 10 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Pulin Che ◽  
Long Cui ◽  
Olaf Kutsch ◽  
Liwang Cui ◽  
Qianjun Li
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Antimalarial Drug ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Screening Assay ◽  
High Throughput Screening Assay

Development of a high-throughput screening assay for drug discovery in SOD1-mediated ALS

2009 ◽  
Vol 65 ◽  
pp. S120
Author(s):  
Gaku Murakami ◽  
Haruhisa Inoue ◽  
Kayoko Tsukita ◽  
Yasuyuki Asai ◽  
Kazuhiro Aiba ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

A high-throughput screening assay for drug discovery in SOD1-mediated ALS targeting the transcription of SOD1

2010 ◽  
Vol 68 ◽  
pp. e311
Author(s):  
Gaku Murakami ◽  
Haruhisa Inoue ◽  
Kayoko Tsukita ◽  
Yasuyuki Asai ◽  
Kazuhiro Aiba ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Filip Zmuda ◽  
Lalitha Sastry ◽  
Sharon M. Shepherd ◽  
Deuan Jones ◽  
Alison Scott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Space ◽  
Protozoan Parasite ◽  
Current Treatment ◽  
Screening Assay ◽  
Content Type ◽  
High Throughput Screening Assay

ABSTRACT Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.

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 Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction

2019 ◽  
Vol 25 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Timothy Marlowe ◽  
Carlos Alvarado ◽  
Andrew Rivera ◽  
Felicia Lenzo ◽  
Rohini Nott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Focal Adhesion ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Drug Target ◽  
Scaffolding Protein ◽  
Cancer Drug ◽  
Screening Assay ◽  
High Throughput Screening Assay

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

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