Optimizations of a novel fluorescence polarization-based high-throughput screening assay for β-catenin/LEF1 interaction inhibitors

Chen, Yun-Yu; Fu, Zhenghao; Li, Dongsheng; Yu-huan, Yue; Li, Xiaoping

doi:10.1016/j.ab.2020.113966

Cited by 8 publications

(16 citation statements)

References 29 publications

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“…Because of the inadequacy of existing HTS methods, we first developed a novel sandwich-like FP screening assay for the rapid discovery of antiviral agents targeting Mpro. FP technique has been widely utilized to identify antagonists targeting protein-protein interactions in drug discovery field [ 23 ]. In a FP screening assay, the change of millipolarization unit (mP) value is dependent on the molecular weight of fluorescent moiety provided that temperature and viscosity remain constant.…”

Section: Resultsmentioning

confidence: 99%

“…The statistical parameter Z factor, which is commonly used to assess the total quality of a HTS method, was determined in a black 384-well microplate. In general, only HTS assays with Z factor of 0.5 or higher are suitable for screening, and the assays with smaller Z factor are required to be further optimized [ 23 ]. To assess this FP screening assay, 1 µM of GC-376 was used to serve as a positive control for quality assessment.…”

Section: Resultsmentioning

confidence: 99%

“…Surface plasmon resonance (SPR) technology has been widely used in assessing the binding affinity between bioactive compound and target protein, and has been regarded as a conventional biochemical method in the analysis of analyte binding affinity [ 23 , 27 ]. The real-time SPR binding kinetics showed that dieckol significantly increased the real refractive index unit (RIU) response in a dose-dependent manner, and the dissociation constant ( K D ) was recorded as 0.22 µM, indicating a strong interaction between dieckol and Mpro in vitro (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The homogenous and highly sensitive FP technique has been widely used in drug discovery, analytical biochemistry, measurement of biomolecular activity and disease diagnosis [ 23 , 29 – 31 ]. This powerful approach is based on a fact that a positive alteration in apparent molecular weight of a fluorescent moiety in solution is indicated by an increasing change of mP value in a FP assay.…”

Section: Discussionmentioning

confidence: 99%

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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

Gao

Lin

et al. 2021

Cell Biosci

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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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

Gao

Lin

et al. 2021

Cell Biosci

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“…It can efficiently find ligands with strong binding to target proteins from complex mixtures [ 153 ]. Fluorescence polarization assay is mainly based on the principle of positive correlation between the intensity of polarized fluorescence and the size of fluorescent molecules, which is widely used in high-throughput drug screening and drug analysis [ 154 ]. It should be noted that each of the above screening techniques has its own scope of application, and a single technique often has certain limitations in accuracy or efficiency.…”

Section: Discussionmentioning

confidence: 99%

Screening S protein – ACE2 blockers from natural products: Strategies and advances in the discovery of potential inhibitors of COVID-19

Liu

et al. 2021

European Journal of Medicinal Chemistry

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The Coronavirus disease, 2019 (COVID-19) is caused by severe acute respiratory syndrome Coronavirus 2 (SARS‐CoV‐2), which poses a major threat to human life and health. Given its continued development, limiting the spread of COVID-19 in the population remains a challenging task. Currently, multiple therapies are being tried around the world to deal with SARS-CoV-2 infection, and a variety of studies have shown that natural products have a significant effect on COVID-19 patients. The combination of SARS-CoV-2 S protein with Angiotensin converting enzyme II(ACE2) of host cell to promote membrane fusion is an initial critical step for SARS-CoV-2 infection. Therefore, screening natural products that inhibit the binding of SARS-CoV-2 S protein and ACE2 also provides a feasible strategy for the treatment of COVID-19. Establishment of high throughput screening model is an important basis and key technology for screening S protein-ACE2 blockers. Based on this, the molecular structures of SARS-CoV-2 and ACE2 and their processes in the life cycle of SARS-CoV-2 and host cell infection were firstly reviewed in this paper, with emphasis on the methods and techniques of screening S protein-ACE2 blockers, including Virtual Screening (VS), Surface Plasmon Resonance (SPR), Biochromatography, Biotin-avidin with Enzyme-linked Immunosorbent assay and Gene Chip Technology. Furthermore, the technical principle, advantages and disadvantages and application scope were further elaborated. Combined with the application of the above screening technologies in S protein-ACE2 blockers, a variety of natural products, such as flavonoids, terpenoids, phenols, alkaloids, were summarized, which could be used as S protein-ACE2 blockers, in order to provide ideas for the efficient discovery of S protein-ACE2 blockers from natural sources and contribute to the development of broad-spectrum anti coronavirus drugs.

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Improved fluorescence‐based assay for rapid screening and evaluation of SARS‐CoV‐2 main protease inhibitors

Zhang,

Yan,

Zhou

et al. 2024

Journal of Medical Virology

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The outbreak of coronavirus disease 2019 (COVID‐19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a global threat to human health. In parallel with vaccines, efficacious antivirals are urgently needed. SARS‐CoV‐2 main protease (Mpro) is an attractive drug target for antiviral development owing to its key roles in virus replication and host immune evasion. Due to the limitations of currently available methods, the development of novel high‐throughput screening assays is of the highest importance for the discovery of Mpro inhibitors. In this study, we first developed an improved fluorescence‐based assay for rapid screening of Mpro inhibitors from an anti‐infection compound library using a versatile dimerization‐dependent red fluorescent protein (ddRFP) biosensor. Utilizing this assay, we identified MG‐101 as a competitive Mpro inhibitor in vitro. Moreover, our results revealed that ensitrelvir is a potent Mpro inhibitor, but baicalein, chloroquine, ebselen, echinatin, and silibinin are not. Therefore, this robust ddRFP assay provides a faithful avenue for rapid screening and evaluation of Mpro inhibitors to fight against COVID‐19.

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Optimizations of a novel fluorescence polarization-based high-throughput screening assay for β-catenin/LEF1 interaction inhibitors

Cited by 8 publications

References 29 publications

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

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

Screening S protein – ACE2 blockers from natural products: Strategies and advances in the discovery of potential inhibitors of COVID-19

Improved fluorescence‐based assay for rapid screening and evaluation of SARS‐CoV‐2 main protease inhibitors

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