scholarly journals A Robust Multiplex Mass Spectrometric Assay for Screening Small-Molecule Inhibitors of CD73 with Diverse Inhibition Modalities

2018 ◽  
Vol 23 (3) ◽  
pp. 264-273 ◽  
Author(s):  
Jessica McManus ◽  
Timothy He ◽  
Julie-Ann Gavigan ◽  
Ghislaine Marchand ◽  
Stephanie Vougier ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Elevated Concentration ◽  
Antitumor Effects ◽  
Extracellular Adenosine ◽  
Biochemical Assays ◽  
Small Molecule Libraries

CD73/Ecto-5′-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire–tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40–50 min.

scholarly journals Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

2011 ◽  
Vol 17 (4) ◽  
pp. 435-447 ◽  
Author(s):  
Daigo Inoyama ◽  
Yu Chen ◽  
Xinyi Huang ◽  
Lesa J. Beamer ◽  
Ah-Ng Tony Kong ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Antioxidant Response ◽  
Binding Affinities ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Peptide Amide

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

scholarly journals High-Throughput Screening of Small Molecule Libraries using SAMDI Mass Spectrometry

2011 ◽  
Vol 13 (4) ◽  
pp. 347-350 ◽  
Author(s):  
Zachary A. Gurard-Levin ◽  
Michael D. Scholle ◽  
Adam H. Eisenberg ◽  
Milan Mrksich
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Libraries

scholarly journals Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach

2013 ◽  
Vol 170 (1) ◽  
pp. 132-140 ◽  
Author(s):  
Margaret R. Duffy ◽  
Alan L. Parker ◽  
Eric R. Kalkman ◽  
Katie White ◽  
Dmytro Kovalskyy ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Screening Approach

scholarly journals High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation

2007 ◽  
Vol 358 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Alex Crowe ◽  
Carlo Ballatore ◽  
Edward Hyde ◽  
John Q. Trojanowski ◽  
Virginia M.-Y. Lee
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Fibril Formation

High throughput screening of small molecule inhibitors of human alternative pathway complement

2010 ◽  
Vol 47 (13) ◽  
pp. 2283-2283
Author(s):  
Yuko Kimura ◽  
Chun-Hao Chiu ◽  
Andrew D. Napper ◽  
Scott L. Diamond ◽  
Wen-Chao Song
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Alternative Pathway

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.

Sign in / Sign up

Export Citation Format

Share Document