scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

scholarly journals High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase

2004 ◽  
Vol 9 (5) ◽  
pp. 391-397 ◽  
Author(s):  
Chongbo Sun ◽  
Yvette Newbatt ◽  
Leon Douglas ◽  
Paul Workman ◽  
Wynne Aherne ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Tumor Development ◽  
Screening Assay ◽  
Amino Terminal ◽  
Terminal Domain ◽  
High Throughput Screening Assay ◽  
Amino Terminal Domain

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, γ-33P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate® to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z′ factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

scholarly journals An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication

2017 ◽  
Vol 292 (30) ◽  
pp. 12577-12588 ◽  
Author(s):  
Subhanita Ghosh ◽  
Abhinav Kaushik ◽  
Sachin Khurana ◽  
Aditi Varshney ◽  
Avishek Kumar Singh ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
B Virus

scholarly journals A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e90766 ◽  
Author(s):  
Nisan Bhattacharyya ◽  
Xin Hu ◽  
Catherine Z. Chen ◽  
Lesley A. Mathews Griner ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Assay System ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals High-Throughput Screening for Small-Molecule Inhibitors of Plasmodium falciparum Glucose-6-Phosphate Dehydrogenase 6-Phosphogluconolactonase

2012 ◽  
Vol 17 (6) ◽  
pp. 738-751 ◽  
Author(s):  
Janina Preuss ◽  
Michael Hedrick ◽  
Eduard Sergienko ◽  
Anthony Pinkerton ◽  
Arianna Mangravita-Novo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Antimalarial Drugs ◽  
Screening Assay ◽  
Compound Libraries ◽  
High Throughput Screening Assay ◽  
Glucose 6 Phosphate Dehydrogenase

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites’ pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries—namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC50 values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.

scholarly journals High-Throughput Screening Assay to Identify Small Molecule Inhibitors of Marburg Virus VP40 Protein

2020 ◽  
Vol 6 (10) ◽  
pp. 2783-2799
Author(s):  
Priya Luthra ◽  
Manu Anantpadma ◽  
Sampriti De ◽  
Julien Sourimant ◽  
Robert A. Davey ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Marburg Virus ◽  
Screening Assay ◽  
High Throughput Screening Assay

Optimization of a High-Throughput Cell-Based Screening Strategy to Identify Small-Molecule Inhibitors of IL-23 Signaling

2020 ◽  
Vol 26 (1) ◽  
pp. 122-129
Author(s):  
Teena M. Varghese ◽  
Paul L. Dudas ◽  
Samantha J. Allen ◽  
Jonathan E. Schneeweis ◽  
Michael F. A. Finley
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Screening Strategy ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Screening Assay ◽  
Gene Assay ◽  
Ifn Γ

Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rβ1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z′ > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose–response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors.

Sign in / Sign up

Export Citation Format

Share Document