scholarly journals Development of a Plasmepsin II Fluorescence Polarization Assay Suitable for High Throughput Antimalarial Drug Discovery

2002 ◽  
Vol 7 (4) ◽  
pp. 367-371 ◽  
Author(s):  
Horst Flotow ◽  
Chung-Yan Leong ◽  
Antony D. Buss
Keyword(s):  
Natural Products ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rapid Screening ◽  
Screening Assay ◽  
Plasmepsin Ii ◽  
Fluorescence Polarization Assay

Despite decades of research, malaria remains the world's most deadly parasitic disease. New treatments with novel mechanisms of action are urgently needed. Plasmepsin II is an aspartyl protease that has been validated as an antimalarial therapeutic target enzyme. Although natural products form the basis of most modern antimalarial drugs, no systematic high-throughput screening has been reported against this target. We have designed an effective strategy for carrying out high-throughput screening of an extensive library of natural products that uses a fluorescence resonance energy transfer primary screening assay in tandem with a fluorescence polarization assay. This strategy allows rapid screening of the library coupled with effective discrimination and elimination of false-positive samples and selection of true hits for chemical isolation of inhibitors of plasmepsin II.

scholarly journals High-Throughput Fluorescence Polarization Assay for Chemical Library Screening against Anti-Apoptotic Bcl-2 Family Member Bfl-1

2011 ◽  
Vol 17 (3) ◽  
pp. 350-360 ◽  
Author(s):  
Dayong Zhai ◽  
Paulo Godoi ◽  
Eduard Sergienko ◽  
Russell Dahl ◽  
Xochella Chan ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Response Analysis ◽  
Chemical Library ◽  
Time Resolved ◽  
Chemical Inhibitors ◽  
Fluorescence Polarization Assay

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

scholarly journals Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gangan Yan ◽  
Dongsheng Li ◽  
Yuan Lin ◽  
Zhenghao Fu ◽  
Haiyan Qi ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Antiviral Agents ◽  
Competitive Inhibitor ◽  
Rapid Screening ◽  
Screening Assay ◽  
Hydrogen Bond Interactions ◽  
Main Protease ◽  
Fluorescence Polarization Assay

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. Results In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. Conclusions This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

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 Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase

2018 ◽  
Vol 23 (9) ◽  
pp. 974-981
Author(s):  
Yu-Chi Juang ◽  
Xavier Fradera ◽  
Yongxin Han ◽  
Anthony William Partridge
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Histidine Decarboxylase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gastric Ulcers ◽  
Statistical Parameters ◽  
Time Resolved ◽  
Neurological Response

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z′ score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure–activity relationships.

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