Late 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 activators
Name: Late 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 activators. ..more
BioActive Compound: 1
Depositor Specified Assays
Source (MLPCN 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 Probe Production Centers Network (MLPCN)
Grant Proposal Number: 5R21NS056337-02
Grant Proposal PI: Richard Morimoto
External Assay ID: HSP70_AG_Lumi_1536_3XEC50 MDRUN
Name: Late 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 activators.
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.
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.
late stage, powders, purchased, synthesized, MDRUN, medchem, SAR, dose response, 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.
The purpose of this assay is to determine dose response curves of synthesized analogues of lead compounds previously 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)" (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 uM.
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 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 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 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 ) )
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
PubChem Activity Outcome and Score:
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 uM 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.
If a compound was active, then it was given a score of 100. If a compound was inactive, it was given a score of 0.
The PubChem Activity Score range for active compounds is 100-100 and for inactive compounds 0-0.
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)
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. Not all compounds requested were available from the MLSMR
** Test Concentration.
Data Table (Concise)