+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4



High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4



Journal of Biomolecular Screening 16(2): 174-182



Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 053552386

Download citation: RISBibTeXText

PMID: 21245471

DOI: 10.1177/1087057110392996


Related references

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2). European Journal of Pharmaceutical Sciences 118: 67-79, 2018

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2). European Journal of Pharmaceutical Sciences 118: 67-79, 2018

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1. Analytical Biochemistry 352(1): 135-141, 2006

High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay. Journal of Biomolecular Screening 14(2): 161-172, 2009

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity. Analytical Biochemistry 440(1): 71-77, 2013

Development and implementation of a miniaturized high-throughput time-resolved fluorescence energy transfer assay to identify small molecule inhibitors of polo-like kinase 1. Assay and Drug Development Technologies 5(6): 723-735, 2008

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay. Journal of Biomolecular Screening 13(10): 1025-1034, 2008

A high throughput screening assay system for the identification of small molecule inhibitors of gsp. Plos One 9(3): E90766-E90766, 2015

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors. Analytical Biochemistry 413(2): 90-96, 2011

A Unique High-Throughput Assay to Identify Novel Small Molecule Inhibitors of Chemotaxis and Migration. Current Protocols in Cell Biology 74: 12.11.1-12.11.13, 2017

Small-molecule collection and high-throughput colorimetric assay to identify PARP1 inhibitors. Methods in Molecular Biology 780: 491-516, 2012

Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2. Slas Discovery 2018: 2472555218817057-2472555218817057, 2018

High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function. Journal of Biomolecular Screening 17(6): 822-828, 2012

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors. Journal of Biomolecular Screening 13(7): 665-673, 2008

A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly. Nucleic Acids Research 32(9): 2707-2715, 2004