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 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 Method for Identification of FKBP12 Ligands

2002 ◽  
Vol 7 (6) ◽  
pp. 526-530 ◽  
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.

scholarly journals Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

2020 ◽  
Vol 7 ◽  
Author(s):  
Fuxiao Liu ◽  
Qianqian Wang ◽  
Yilan Huang ◽  
Ning Wang ◽  
Youming Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Rapid Screening ◽  
Canine Distemper ◽  
Virus Propagation ◽  
The Family ◽  
Conventional Methods

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

Closing the empty anti-Babesia gibsoni drug pipeline in vitro using fluorescence-based high throughput screening assay

2020 ◽  
Vol 75 ◽  
pp. 102054 ◽  
Author(s):  
Mohamed Abdo Rizk ◽  
Shengwei Ji ◽  
Mingming Liu ◽  
Shimaa Abd El-Salam El-Sayed ◽  
Yongchang Li ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Babesia Gibsoni ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Drug Pipeline

Respirometric Screening and Characterization of Mitochondrial Toxicants Within the ToxCast Phase I and II Chemical Libraries

2020 ◽  
Vol 176 (1) ◽  
pp. 175-192
Author(s):  
Daniel R Hallinger ◽  
Hayley B Lindsay ◽  
Katie Paul Friedman ◽  
Danielle A Suarez ◽  
Steven O Simmons
Keyword(s):  
High Throughput Screening ◽  
Predictive Accuracy ◽  
Electron Flow ◽  
Mitochondrial Toxicity ◽  
Toxicity Screening ◽  
Screening Assay ◽  
Adverse Health Outcomes ◽  
Chemically Induced ◽  
Transport Chain

Abstract Mitochondrial toxicity drives several adverse health outcomes. Current high-throughput screening assays for chemically induced mitochondrial toxicity typically measure changes to mitochondrial structure and may not detect known mitochondrial toxicants. We adapted a respirometric screening assay (RSA) measuring mitochondrial function to screen ToxCast chemicals in HepG2 cells using a tiered testing strategy. Of 1042 chemicals initially screened at a singlemaximal concentration, 243 actives were identified and rescreened at 7 concentrations. Concentration-response data for 3 respiration phases confirmed activity and indicated a mechanism for 193 mitochondrial toxicants: 149 electron transport chain inhibitors (ETCi), 15 uncouplers and 29 adenosine triphosphate synthase inhibitors. Subsequently, an electron flow assay was used to identify the target complex for 84 of the 149 ETCi. Sixty reference chemicals were used to compare the RSA to existing ToxCast and Tox21 mitochondrial toxicity assays. The RSA was most predictive (accuracy = 90%) of mitochondrial toxicity. The Tox21 mitochondrial membrane potential assay was also highly predictive (accuracy = 87%) of bioactivity but underestimated the potency of well-known ETCi and provided no mechanistic information. The tiered RSA approach accurately identifies and characterizes mitochondrial toxicants acting through diverse mechanisms and at a throughput sufficient to screen large chemical inventories. The electron flow assay provides additional confirmation and detailed mechanistic understanding for ETCi, the most common type of mitochondrial toxicants among ToxCast chemicals. The mitochondrial toxicity screening approach described herein may inform hazard assessment and the in vitro bioactive concentrations used to derive relevant doses for screening level chemical assessment using new approach methodologies.

scholarly journals High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor

2019 ◽  
Vol 24 (9) ◽  
pp. 915-927
Author(s):  
P. Heine ◽  
G. Witt ◽  
A. Gilardi ◽  
P. Gribbon ◽  
L. Kummer ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Binding Pocket ◽  
G Protein Coupled Receptor ◽  
Library Screen ◽  
A Cell ◽  
Fluorescence Polarization Assay ◽  
G Protein Coupled

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.

scholarly journals A Novel High-Throughput Screening Assay for Sickle Cell Disease Drug Discovery

2009 ◽  
Vol 14 (4) ◽  
pp. 330-336 ◽  
Author(s):  
Eszter Pais ◽  
John S. Cambridge ◽  
Cage S. Johnson ◽  
Herbert J. Meiselman ◽  
Timothy C. Fisher ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rapid Screening ◽  
Cell Disease ◽  
Screening Assay ◽  
Drug Candidates ◽  
Test Molecule ◽  
High Throughput Screening Assay

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. ( Journal of Biomolecular Screening. 2009:330-336)

scholarly journals Current Trends in SPR Biosensing of SARS-CoV-2 Entry Inhibitors

Chemosensors ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 330
Author(s):  
Elba Mauriz ◽  
Laura M. Lechuga
Keyword(s):  
High Throughput Screening ◽  
Antiviral Agents ◽  
Host Cells ◽  
Binding Affinities ◽  
Entry Inhibitors ◽  
Antiviral Compounds ◽  
Current Trends ◽  
Main Protease ◽  
Emerging Risk ◽  
Repurposed Drugs

The emerging risk of viral diseases has triggered the search for preventive and therapeutic agents. Since the beginning of the COVID-19 pandemic, greater efforts have been devoted to investigating virus entry mechanisms into host cells. The feasibility of plasmonic sensing technologies for screening interactions of small molecules in real time, while providing the pharmacokinetic drug profiling of potential antiviral compounds, offers an advantageous approach over other biophysical methods. This review summarizes recent advancements in the drug discovery process of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) inhibitors using Surface Plasmon Resonance (SPR) biosensors. A variety of SPR assay formats are discussed according to the binding kinetics and drug efficacies of both natural products and repurposed drugs. Special attention has been given to the targeting of antiviral agents that block the receptor binding domain of the spike protein (RBD-S) and the main protease (3CLpro) of SARS-CoV-2. The functionality of plasmonic biosensors for high-throughput screening of entry virus inhibitors was also reviewed taking into account experimental parameters (binding affinities, selectivity, stability), potential limitations and future applications.

scholarly journals Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies

2020 ◽  
Author(s):  
Mikolaj Zmudzinski ◽  
Wioletta Rut ◽  
Kamila Olech ◽  
Jarosław Granda ◽  
Mirosław Giurg ◽  
...  
Keyword(s):  
Small Molecule ◽  
Antiviral Agents ◽  
Small Molecule Drug ◽  
Main Protease ◽  
Promising Target ◽  
Viral Survival ◽  
Dual Inhibitors ◽  
Viral Polyprotein ◽  
Covalent Inhibitor

AbstractProteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage - a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and anti-viral assays. In this study, we screened a collection of 23 ebselen derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.

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