Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

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High-Throughput Fluorescence Polarization Method for Identification of FKBP12 Ligands

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057102238626 ◽

2002 ◽

Vol 7 (6) ◽

pp. 526-530 ◽

Cited By ~ 36

Author(s):

S. Bollini ◽

J. J. Herbst ◽

G. T. Gaughan ◽

T. A. Verdoorn ◽

J. Ditta ◽

...

Keyword(s):

High Throughput ◽

Fluorescence Polarization ◽

High Throughput Screening ◽

Metal Ion ◽

Gel Filtration ◽

Immunosuppressive Drug ◽

Fluorescence Polarization Assay ◽

High Throughput Screens

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His6-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with Ki values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

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A high‐throughput fluorescence polarization assay for the determination of Kd for soluble epoxide hydrolase inhibitors

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.479.21 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Richard H. Ingraham ◽

Rachel R. Kroe ◽

Maurice M. Morelock ◽

John R. Proudfoot ◽

Anne B. Eldrup ◽

...

Keyword(s):

High Throughput ◽

Fluorescence Polarization ◽

Epoxide Hydrolase ◽

Soluble Epoxide Hydrolase ◽

Soluble Epoxide Hydrolase Inhibitors ◽

Fluorescence Polarization Assay

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Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2020.113182 ◽

2020 ◽

Vol 184 ◽

pp. 113182 ◽

Cited By ~ 1

Author(s):

Pimyupa Manaswiyoungkul ◽

Fettah Erdogan ◽

Olasunkanmi O. Olaoye ◽

Aaron D. Cabral ◽

Elvin D. de Araujo ◽

...

Keyword(s):

Dna Binding ◽

High Throughput ◽

Fluorescence Polarization ◽

Binding Domain ◽

Dna Binding Domain ◽

Fluorescence Polarization Assay

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Development and Application of a High-Throughput Fluorescence Polarization Assay to Target Pim Kinases

Assay and Drug Development Technologies ◽

10.1089/adt.2015.685 ◽

2016 ◽

Vol 14 (1) ◽

pp. 50-57 ◽

Cited By ~ 2

Author(s):

Seongho Lee ◽

Victor Sukbong Hong

Keyword(s):

High Throughput ◽

Fluorescence Polarization ◽

Pim Kinases ◽

Fluorescence Polarization Assay

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A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/138620708784534770 ◽

2008 ◽

Vol 11 (5) ◽

pp. 396-409 ◽

Cited By ~ 23

Author(s):

Adam Kimple ◽

Adam Yasgar ◽

Mark Hughes ◽

Ajit Jadhav ◽

Francis Willard ◽

...

Keyword(s):

High Throughput ◽

Fluorescence Polarization ◽

Fluorescence Polarization Assay ◽

Goloco Motif

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

Cell & Bioscience ◽

10.1186/s13578-021-00720-3 ◽

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.

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An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006893117 ◽

2020 ◽

Vol 117 (31) ◽

pp. 18431-18438 ◽

Cited By ~ 2

Author(s):

Yao Yao ◽

Rameshwar U. Kadam ◽

Chang-Chun David Lee ◽

Jordan L. Woehl ◽

Nicholas C. Wu ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

Fluorescence Polarization ◽

Neutralizing Antibodies ◽

Influenza A ◽

Cyclic Peptide ◽

Host Cells ◽

Vitro Assay ◽

Group 1

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

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