Development and validation of a hedgehog heparin-binding assay for high-throughput screening

The Anti- Wolbachia (A·WOL) consortium at the Liverpool School of Tropical Medicine (LSTM) has partnered with the Global High-Throughput Screening (HTS) Centre at AstraZeneca to create the first anthelmintic HTS for neglected tropical diseases (NTDs). The A·WOL consortium aims to identify novel macrofilaricidal drugs targeting the essential bacterial symbiont ( Wolbachia) of the filarial nematodes causing onchocerciasis and lymphatic filariasis. Working in collaboration, we have validated a robust high-throughput assay capable of identifying compounds that selectively kill Wolbachia over the host insect cell. We describe the development and validation process of this complex, phenotypic high-throughput assay and provide an overview of the primary outputs from screening the AstraZeneca library of 1.3 million compounds.

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Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus

Journal of Virological Methods ◽

10.1016/j.jviromet.2008.01.016 ◽

2008 ◽

Vol 149 (1) ◽

pp. 12-19 ◽

Cited By ~ 25

Author(s):

Mohamad Aljofan ◽

Matteo Porotto ◽

Anne Moscona ◽

Bruce A. Mungall

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Antiviral Drugs ◽

Hendra Virus ◽

Development And Validation

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Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0004094 ◽

2015 ◽

Vol 9 (9) ◽

pp. e0004094 ◽

Cited By ~ 24

Author(s):

Andrea Nühs ◽

Manu De Rycker ◽

Sujatha Manthri ◽

Eamon Comer ◽

Christina A. Scherer ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Leishmania Donovani ◽

Intracellular Activity ◽

Development And Validation ◽

Screening Cascade

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Identification of Influenza Virus Inhibitors Targeting NS1A Utilizing Fluorescence Polarization–Based High-Throughput Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111431488 ◽

2012 ◽

Vol 17 (4) ◽

pp. 448-459 ◽

Cited By ~ 20

Author(s):

Eun Jeong Cho ◽

Shuangluo Xia ◽

Li-Chung Ma ◽

Jon Robertus ◽

Robert M. Krug ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

Fluorescence Polarization ◽

High Throughput Screening ◽

Binding Assay ◽

Nonstructural Protein ◽

Binding Motif ◽

Nonstructural Protein 1 ◽

Dsrna Binding ◽

Ns1a Protein

This article describes the development of a simple and robust fluorescence polarization (FP)–based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z′ score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.

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A molecular toolbox for ADP-ribosyl binding proteins

10.1101/2021.05.31.445082 ◽

2021 ◽

Author(s):

Sven T Sowa ◽

Albert Galera-Prat ◽

Mirko M Maksimainen ◽

Heli I Alanen ◽

Sarah Wazir ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Viral Infections ◽

Binding Assay ◽

Fluorescent Proteins ◽

Protein Fusion ◽

E Coli ◽

Wide Range ◽

Gi Protein ◽

Protein Alpha Subunit

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

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Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111424605 ◽

2011 ◽

Vol 16 (10) ◽

pp. 1206-1216 ◽

Cited By ~ 7

Author(s):

Patricia Haus ◽

Michael Korbus ◽

Michael Schröder ◽

Franz-Josef Meyer-Almes

Keyword(s):

Histone Deacetylase ◽

High Throughput ◽

Fluorescence Anisotropy ◽

Histone Deacetylases ◽

High Throughput Screening ◽

Cell Cycle Progression ◽

Histone Deacetylase Inhibitors ◽

Binding Assay ◽

Cellular Functions ◽

Isoform Selectivity

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.

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A Homogeneous 384-Well High-Throughput Binding Assay for a TNF Receptor Using Alphascreen Technology

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103257804 ◽

2003 ◽

Vol 8 (5) ◽

pp. 522-532 ◽

Cited By ~ 18

Author(s):

Janet Wilson ◽

Claudia Pena Rossi ◽

Susanna Carboni ◽

Christèle Fremaux ◽

Dominique Perrin ◽

...

Keyword(s):

High Throughput ◽

Protein Interactions ◽

High Throughput Screening ◽

Binding Assay ◽

Tumor Necrosis Factor Receptor ◽

Receptor Activation ◽

Affinity Constant ◽

Protein Protein Interactions ◽

Ligand Interaction ◽

High Throughput Assay

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen™ technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation. ( Journal of Biomolecular Screening 2003:522-532)

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