Luminescence Cell-Based Primary HTS to Identify Inhibitors of Heat Shock Factor 1 (HSF1).
Assay Overview: Modified NIH3T3, transformed to express firefly luciferase under the control of a HSF-1 response element, will be exposed to small molecules. After 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After 8hr incubation in the presence of this stressor, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent. ..more
BioActive Compounds: 2328
Depositor Specified Assays
Keywords: Heat Shock Factor-1 (HSF-1), Stress Response, MG132, NIH3T3, Luminescence
Assay Overview: Modified NIH3T3, transformed to express firefly luciferase under the control of a HSF-1 response element, will be exposed to small molecules. After 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After 8hr incubation in the presence of this stressor, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent.
Expected Outcome: Identification of HSF-1 inhibitors in those instances where there is a loss of luminescence signal due to the prevention of HSF-1 being able to drive the expression of the luciferase reporter. Potential false positives in this assay included those compounds which act not by specifically inhibiting HSF-1, but by inhibiting luciferase or are broad spectrum inhibitors of transcription/translation.
HGL HSF-1 luciferase assay:
The NIH3T3-HGL cell line is modified version of NIH3T3 fibroblasts with an integrated eGFP-Firefly luciferase fusion construct under the control of a
Heat shock response element. The NIH3T3-HGL cell line was generously provided for this study by Luke Whitesell.
The HGL cell line is propagated in Opti-MEM (Invitrogen, cat# 31985-088) supplemented with 5% heat inactivated fetal bovine serum (FBS) (Invitrogen, cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, cat# 10378-016) at 37'C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening (HTS) assays, cells are grown in T225 flask (BD Falcon, cat# 353138) or Hyperflasks (Corning, cat# 10010), harvested at more than 80% confluence using Accumax cell detachment solution (Innovative Cell Technologies, cat# AM105). Cell number is counted using a Cellometer Auto M10 cell counter (Nexelcom Bioscience) and viability is measured by mixing one volume of cells with one same volume of Trypan Blue solution (0.4%)(dilution 1/2). Only cultures of >94% viability are utilized for HTS.
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform General system (GS) automation unit:
Day 1 (Cell plating):
1. HGL cells are harvested and re-suspended in Opti-MEM with 2.5% Heat inactivated FBS, 1% penicillin/streptomycin/glutamine. HGL cells (from an initial cell suspension of 200,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, cat# 8867BC) at a final density of 4,000 cells per well in final volume of 20 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plate (cell plate) are placed in Liconic Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37'C in the Liconic CO2 incubator 9 (General automation system (GS)) (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3.The MLPCN test compounds plates are transferred from the Compound Management incubators STX1000#1 and #2 system (Liconic Instruments) on the GS to the Liconic CO2 incubator 8 before the initiation of the pinning run. The In-plate positive control compound plate (sentinel) (25uM Rocaglamide A (RocA), Alexis Biochemicals ALX-350-121-C100), or vehicle (DMSO only) are already present in the Liconic incubator 8 at the beginning of the pinning. Both STX and Liconic 8 CO2 incubator temperature are kept at 20'C. MLPCN test compounds plates are pinned as well as the in-plate positive control (32 wells, 50nM final conc. RocA) are pinned consecutively one after the other and transferred into one assay plate. Each compound plate is pinned into duplicate assay plates. The final concentration for the MLPCN test compounds is 7.5uM with final concentration no more than 1% DMSO.
4. After 30min incubation with the compounds, 50uM MG132 (Enzo, cat# PI-102) in 1X PBS (Invitrogen, cat# 10010) is added in 1uL with the CombinL (Thermo) (2.5uM final conc. MG132) to induce HSF-1. The cells are incubated in the presence of MG132 for a further 8hrs.
(Reading luminescence from assay plates with Envision):
5. After 8 hr incubation, 20uL of Steady-Glo luciferase (Promega, cat# E2550) is added to each assay plate using a MultiDrop Combi (Thermo). Luminescence is measured in each well (0.1 second/well) using the Envision plate reader (Perkin Elmer)(Corning plate setting).
HTS Data Analysis
Each compound was tested in duplicate assay wells; a small percentage of compounds were tested in quadruplicate.
23 neutral control wells (DMSO) were included on every plate.
Active compounds result in decreased readout of luminescence signal.
Analysis used to determine PubChem Activity Score and Outcome
The raw luminescent signal of the DMSO control wells was normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
This normalization was then applied to all compound treated wells, giving an activity score as percent change in luminescence signal relative to the intraplate neutral control.
A plate correction matrix was applied using the 'Run-wise Multiplicative Correction' in Genedata Assay Analyzer (v7.0.3)
The final PUBCHEM_ACTIVITY_SCORE was calculated by multiplying the mean of all valid replicate values (which can be described in units of negative percent activity) was mulitplited by -1, resulting in a score between 0 and 100 (in units of 'percent inhibition').
The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of -50%:
Activity_Outcome = 1 (inactive)
0% of replicates fall outside threshold.
Activity_Outcome = 2 (active)
>50% of replicates fall outside threshold.
Activity_Outcome = 3 (inconclusive)
>0% and <= 50% of replicates fall outside threshold.
Data Table (Concise)