uHTS fluorescent assay for identification of inhibitors of ATG4B
Autophagy is an evolutionarily conserved process whereby cells catabolize damaged proteins and organelles for purposes of generating substrates for sustaining ATP production during times of nutrient deprivation. The autophagic process involves membrane vesicles engulfing cytosol and organelles, delivering their contents to lysosomes for digestion. The genes responsible for autophagy have been more ..
BioActive Compounds: 1734
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH090871-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA
Autophagy is an evolutionarily conserved process whereby cells catabolize damaged proteins and organelles for purposes of generating substrates for sustaining ATP production during times of nutrient deprivation. The autophagic process involves membrane vesicles engulfing cytosol and organelles, delivering their contents to lysosomes for digestion. The genes responsible for autophagy have been identified, largely through genetic analysis of yeast, Saccharomyces cerevisiae, and are conserved in mammals, plants, and essentially all eukaryotes. While autophagy is critical for cell survival in the context of nutrient deprivation, circumstances where autophagy and cell death occur in tandem have been identified, especially in tumor biology. For example, apoptosis-defective tumors are reliant on autophagy to survive metabolic stress. Also, autophagy inhibitors can be used to induce acute necrotic cell death, which may be facilitated by concurrent proteasome inhibition, enabling tumor eradication. In the adjuvant setting, and after elimination of a large proportion of the tumor by radiation and chemotherapy, the remaining cells can reside in a disrupted and stressed environment, susceptible to inhibition of the autophagy survival mechanism. Tumor cells in the process of metastasis can be similarly vulnerable.
Chemical modulators of autophagy are essentially non-existent, with the only available agent, 3-methyladenine, requiring millimolar concentrations to inhibit class III phosphatidyl inositol kinases (PI3Ks) involved in autophagy. A need exists for chemicals that target specific components of the autophagy machinery -- both for use as research tools for addressing questions about the role of autophagy in diseases such as cancer.
Autophagins are a class of cytosolic cysteine proteases required for autophagy. The human genome contains four independent genes encoding the Autophagins, including ATG4A, 4B, 4C, and 4D. These proteases cleave Gly120 site of LC3 (ATG8) to promote its conjugation with phosphatidylethanolamine (PE) by a ubiquitin-like system, which is required for autophagosome formation and that participate in targeting these vesicles to lysosomes for fusion and degradation of their contents. Autophagins also promote deconjugation of LC3-PE to liberate LC3 from membrane at or before the final stage of fusion between autophagosome and lysosomes, suggesting that deconjugation of LC3-PE is required for the fusion.
In this assay, a High Throughput Screening (HTS) assay based on fluorescence intensity is utilized to screen for compounds that inhibit Autophagin 1 (ATG4B). The HTS assay utilizes a cleavable form of Phospholipase A2 (PLA2), which is expressed as a fusion protein with the Autophagin substrate LC3/ATG8 appended to its N-terminus. The addition of sequences to the N-terminus of PLA2 inhibits the activity of this enzyme. Cleavage by proteases removing the N-terminal extension then restores enzyme activity, constituting the basis for a protease assay. Together, these efforts will result in validated chemical probes for studying the autophagy in a variety of biological settings.
A. Brief Description of the Assay:
This assay is to look for inhibitors of ATG4B enzyme that are able to prevent cleavage of LC3-PLA2. It will be measured by Fluorescence in 1536 well plate format.
Item, Source, Cat#
ATG4B Enzyme, Assay Provider
LC3-PLA2, Assay Provider
NBD-C6-HPC, Sigma-Aldrich, N3786)
TRIS-HCl pH 8.0
CaCl2, Sigma-Aldrich, 499609
Dithiothreitol, Sigma-Aldrich, 646563
1536-well Black solid bottom (Normal-Binding) plate, Greiner, 782076
1. Using LabCyte Echo, transfer 5 nL from a 10 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 8.33 uM, 0.5% DMSO).
2. Prepare reagents as stated in D. Recipe and keep on ice.
Note: Reagents should be used as soon as possible. Final signal will degrade by half over 1.5hrs. Reagents are stable once reaction has started.
3. Using the Matrix Combi, add 3ul/well of Mix 2 to col. 1-48.
4. Using the Matrix Combi, add 3ul/well of Mix 3 to col. 1-2 for the positive control.
5. Using the Matrix Combi, add 3ul/well Mix 1 to col. 3-48 for the negative control and test compound wells.
6. Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge.
7. Incubate plates in the dark at room temperature for 1 hour.
8. Read plates using a ViewLux Fluorescence Intensity protocol at 485nm excitation and 535nm emission wavelengths.
Note: Multiple freeze-thaws of ATG4B, LC3-PLA2 and NBD-C6-HPC are not recommended!
20mM TRIS-HCl pH 8.0, 2mM CaCl2, 1mM DTT
0.2nM working (0.1nM FAC) ATG4B, 40uM working (20uM FAC) NBD-C6-HPC, Assay Buffer
200nM working (100nM FAC) LC3-PLA2, Assay Buffer
40uM working (20uM FAC) NBD-C6-HPC, Assay Buffer
Compounds that demonstrated % activity of >= 40 % at 8.33 uM concentration are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)