Cytotoxicity counterscreen assay for transcriptional activators of heat shock protein 70 (Hsp70)
Name: Cytotoxicity counterscreen assay for transcriptional activators of heat shock protein 70 (Hsp70) ..more
BioActive Compounds: 21
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: CYTOX_INH_LUMI_1536_CC50
Name: Cytotoxicity counterscreen assay for transcriptional activators of heat shock protein 70 (Hsp70)
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.
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.
Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, chaperone, HTS, high throughput screen, cytotoxicity, viability, CellTiter-Glo, counterscreen, dose response screen, 1536, reporter gene, transcription, luciferase, luminescence, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
The purpose of this assay is to determine the cytotoxicity of 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) and confirmed activity via dose-response assays entitled, "Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)" (PubChem AID 1252). The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP found in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. Thus, well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that induce cell death will reduce ATP levels, and therefore reduce well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 99 uM. Compounds active in the assays above and inactive in this cytotoxicity counterscreen are considered nontoxic inducers of Hsp70 transcription.
Prior to the start of the assay, HeLa cells were plated at 500 cells per well in 1536-well plates in 5 microliters of growth media (Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% FBS and 1% Pen/Strep/Neo). Plates were incubated for 4 hours at 37C, 5% CO2 and 95% relative humidity. Next, 50 nL of test compounds in DMSO or DMSO alone were added to the sample or control wells, respectively. Plates were then placed back in the incubator for 16 hours. After incubation, 5 ul of CellTiter-Glo reagent were added to each well, and plates were allowed to incubate for 15 minutes at room temperature. Luminescence was recorded for 30 seconds per well using the ViewLux(tm) reader (PerkinElmer, Turku, Finland). Percent cytotoxicity was expressed relative to wells containing media only (100%) and wells containing cells treated with DMSO only (0%).
The percent cytotoxicity for each compound was defined according to the following mathematical formula:
% Cytotoxicity = 100* (Median_Negative_Control - Test_Compound) / (Median_Negative_Control - Median_Positive_Control)
Positive_Control is defined as wells containing media only,
Negative_Control is defined as wells containing cells treated with DMSO,
Test_Compound is defined as wells containing cells treated with test compound.
For each test compound, percent cytotoxicity 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). The reported CC50 values were generated from fitted curves by solving for the X-intercept at the 50% inhibition level of the Y-intercept. In cases where the highest concentration tested did not result in > 50% cytotoxicity or where no curve fit was achieved, the CC50 was determined manually depending on the observed cytotoxicity at the individual concentrations. Compounds with CC50 values greater than 10 uM were considered inactive. Compounds with CC50 values equal to or less than 10 uM were considered active.
Any compound with a percent cytotoxicity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent cytotoxicity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by potency, with the most potent compounds assigned the highest activity scores.
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)
CellTiter-Glo Reagent (Promega, part G7572)
1536-well plates (Greiner, part 789173)
T175 flasks (Corning, part 430828)
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.
The inactive compounds of this assay have activity score range of 0 to 49 and active compounds range of activity score is 51 to 100.
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: HeLa
* Activity Concentration. ** Test Concentration.
Data Table (Concise)