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BioAssay: AID 1252

Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)

Name: Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70) ..more
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 Tested Compounds
 Tested Compounds
All(72)
 
 
Active(12)
 
 
Inactive(60)
 
 
 Tested Substances
 Tested Substances
All(72)
 
 
Active(12)
 
 
Inactive(60)
 
 
AID: 1252
Data Source: The Scripps Research Institute Molecular Screening Center (HSP70_AG_Lumi_1536_3X % Act)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-04-17
Modify Date: 2008-08-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 12
Depositor Specified Assays
AIDNameTypeComment
588815Summary of the probe development efforts to identify transcriptional activators of heat shock protein 70 (Hsp70).summary
651950Late stage assay for the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70). Luminescence-based-cell-based high-throughput dose response assay for Hsp70 activatorsother
1259Cytotoxicity counterscreen assay for transcriptional activators of heat shock protein 70 (Hsp70)confirmatory
651945Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess Hsp70 gene expression changesother
651948Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): chemiluminescence-based biochemical western blot assay for transcriptional activators of heat shock protein Hsp70other
651964Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess gene expression changes in downstream target genesother
651963Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based cell-based microscopic assay to assess aggregation of polyQ35 in C. elegans larvaeother
651992Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): yellow fluorescent protein (YFP) quenching assay to assess changes in trafficking of DeltaF508-CFTRother
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Richard Morimoto, Northwestern University
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 5 R21 NS056337-02
Grant Proposal PI: Richard Morimoto

External Assay ID:HSP70_AG_Lumi_1536_3X % Act

Name: Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)

Description:

The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival (1, 2). Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.

A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response (3, 4). Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells (5). Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins (6-8). High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.


References:

1.Gupta, R.S., and Singh, B. 1994. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. Curr Biol 4:1104-1114.
2.Lindquist, S., and Craig, E.A. 1988. The heat-shock proteins. Annu Rev Genet 22:631-677.
3.Satyal, S.H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J.M., and Morimoto, R.I. 2000. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 97:5750-5755.
4.Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J., and Rubinsztein, D.C. 2000. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci U S A 97:2898-2903.
5.Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., and Zoghbi, H.Y. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148-154.
6.Krobitsch, S., and Lindquist, S. 2000. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A 97:1589-1594.
7.Kazemi-Esfarjani, P., and Benzer, S. 2000. Genetic suppression of polyglutamine toxicity in Drosophila. Science 287:1837-1840.
8.Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L., and Bonini, N.M. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat Genet 23:425-428.
9. Westerheide S.D., Bosman J.D., Mbadugha B.N., Kawahara T.L., Matsumoto G., Kim S., Gu W., Devlin J.P., Silverman R.B., Morimoto R.I. 2004. Celastrols as Inducers of the Heat Shock Response and Cytoprotection. JBC 279:56053-56060.


Keywords:

Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, chaperone, agonist, activator, HTS, high throughput screen, dose response screen, 1536, reporter gene, transcription, luciferase, luminescence, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
Protocol
Assay Overview:

The purpose of this assay is to determine dose response curves for test compounds from the MLSCN library identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)" (PubChem AID 1203). In this assay induction of the heat shock response by test compound is measured in a HeLa cell line stably expressing a luciferase reporter under control of the human Hsp70 promoter. As designed, a compound that activates the Hsp70 promoter will increase luciferase transcription, and thus increase well luminescence. Compounds were tested in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 100 micromolar.

Protocol Summary:

The hsp70.1pr-luc HeLa cell line was routinely cultured in tissue culture flasks in growth media (Dulbecco's Modified Eagle's Media supplemented with 10% v/v fetal bovine serum, 1% pen-strep-neomycin antibiotic mixture and 1% Geneticin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).

Prior to the start of the assay, cells were resuspended in growth media at a concentration of 750,000 cells/mL. Next, 5 ul of cell suspension were dispensed into each well of 1536-well plates (3,750 cells per well). After incubation for 4 hours at 37 degrees C, 5% CO2 and 95% RH, the assay was started by dispensing 50 nL of test compound in DMSO to sample wells, DMSO alone (1% final concentration) to negative control wells, or MG132 (final nominal EC100 concentration of 30 uM, set as 100% activation) to positive control wells. The plates were then incubated for 16 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader.

The percent activity was defined using the following mathematical formula:

% Activity = 100*((Test_Compound - Median_Low_Control) / (Median_High_Control - Median_Low_Control))

Where:
Test_Compound is defined as wells containing test compound
High_Control is defined as wells containing MG132
and Low_Control is define as wells containing DMSO

For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems).

A modified method was used to determine whether compounds were active or inactive (see Comment section below). Compounds with >50% activation at concentrations lower than or equal to 11 micromolar were considered active and assigned an activity score of hundred. Compounds with <50% activation at all test concentrations less than 11 uM were assigned an activity score of zero.


List of reagents:

Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092)
Fetal Bovine Serum (Hyclone, part SH 30088.03)
Geneticin (Invitrogen, part 10131-027)
Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)
1536-well plates (Greiner, part 789173)
HYPERflasks (Corning, part 10010)
Reference agonist MG132 (American Peptide, part 81-5-15)
Comment
Instead of graded, sigmoidal dose-response curves, the dose-response profiles of all compounds assayed were characterized by an increasing luminescence response with increasing test compound dose, followed by a sharp decrease in response at higher dose concentrations. This type of dose-response profile has been previously reported for this cell line (9) Because no compounds exhibited an activation profile which could be fit to our sigmoidal dose-response curve algorithm (described above), EC50 values could not be determined for compounds tested in this assay.

Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate luciferase activity directly, and compounds that quench or emit luminescence in the well. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50*The concentration at which 50 percent of the activity in the agonist assay is observed; (EC50) shown in micromolar.
data for each compound.
FloatμM
2Number of DataPointsOverall number of data points of normalized percent Activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
3Activation at 5.0 nM (0.005μM**)Value of %Activation at 5 nanomolar activator concentration; average of triplicate measurement.Float%
4Activation at 15.1 nM (0.0151μM**)Value of %Activation at 15 nanomolar activator concentration; average of triplicate measurementFloat%
5Activation at 45.3 nM (0.0453μM**)Value of %Activation at 45 nanomolar activator concentration; average of triplicate measurementFloat%
6Activation at 135.8 nM (0.1358μM**)Value of %Activation at 135 nanomolar activator concentration; average of triplicate measurementFloat%
7Activation at 407.4 nM (0.4074μM**)Value of %Activation at 407 nanomolar activator concentration; average of triplicate measurementFloat%
8Activation at 1.2 uM (1.2μM**)Value of %Activation at 1.2 micromolar activator concentration; average of triplicate measurement.Float%
9Activation at 3.7 uM (3.7μM**)Value of %Activation at 3.7 micromolar activator concentration; average of triplicate measurementFloat%
10Activation at 11.0 uM (11μM**)Value of %Activation at 11 micromolar activator concentration; average of triplicate measurement.Float%
11Activation at 33.0 uM (33μM**)Value of %Activation at 33 micromolar activator concentration; average of triplicate measurement.Float%
12Activation at 99.0 uM (99μM**)Value of %Activation at 99 micromolar activator concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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