scholarly journals Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors

2014 ◽  
Vol 19 (7) ◽  
pp. 1024-1034 ◽  
Author(s):  
Dariusz Pytel ◽  
Kathleen Seyb ◽  
Min Liu ◽  
Soumya S. Ray ◽  
John Concannon ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Quality ◽  
Protein Translation ◽  
Enzymatic Characterization ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Stress Dependent

PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the Km of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer–based assay that yielded a robust Z′ factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

2011 ◽  
Vol 413 (2) ◽  
pp. 90-96 ◽  
Author(s):  
Kenneth Segers ◽  
Hugo Klaassen ◽  
Anastasios Economou ◽  
Patrick Chaltin ◽  
Jozef Anné
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

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 SCAN ™—A High-Throughput Assay for Detecting Small Molecule Binding to RNA Targets

2009 ◽  
Vol 14 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Chris Baugh ◽  
Shaohui Wang ◽  
Bin Li ◽  
James R. Appleman ◽  
Peggy A. Thompson
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
Laboratory Equipment ◽  
Rna Targets ◽  
High Throughput Screening Assay ◽  
Small Molecule Ligands ◽  
Hcv Ires ◽  
High Throughput Assay

A novel optical-based high-throughput screening technology has been developed for increasing the rate of discovering chemical leads against RNA targets. SCAN™ ( Screen for Compounds with Affinity for Nucleic Acids) is an affinity-based assay that identifies small molecules that bind and recognize structured RNA elements. This technology provides the opportunity to conduct high-throughput screening of a new class of targets—RNA. SCAN™ offers many attractive features including a simple homogeneous format, low screening costs, and the ability to use common laboratory equipment. A SCAN™ assay was developed for the HCV IRES Loop IIId RNA domain. A high-throughput screen of our entire compound library resulted in the identification of small molecule ligands that bind to Loop IIId. The Z′ values were greater than 0.8, showing this to be a robust high-throughput screening assay. A correlation between SCAN™ EC50 and KD values is reported suggesting the ability to use the assay for compound optimization. ( Journal of Biomolecular Screening 2009:219-229)

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

A serendipitously identified novel small molecule procoagulant compound giving rise to a high-throughput screening assay based on human plasma

2013 ◽  
Vol 132 (2) ◽  
pp. 248-255 ◽  
Author(s):  
David Gustafsson ◽  
Sofi Nielsen ◽  
Jane McPheat ◽  
Fredrik Wågberg ◽  
Karin Kaspersson ◽  
...  
Keyword(s):  
Human Plasma ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document