AID	Name	Type	Comment
2098	Luminescence Cell-Based Primary HTS to Identify Inhibitors of Heat Shock Factor 1 (HSF1).	screening	Primary HTS
2118	Identifying Small Molecules To Probe the Role of Heat Shock Factor 1 in Cancer	summary	Project Summary

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 37C 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 μL. 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 37C 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) (25μM 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 20C. 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 7.5μM with final concentration no more than 1% DMSO.

4. After 30min incubation with the compounds, 50M MG132 (Enzo, cat PI-102) in 1X PBS (Invitrogen, cat 10010) is added in 1L 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, 20L 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).

Data Analysis:

Neutral control (NC) wells were included on every plate.
Active inhibitor compounds result in decreased signal.

The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
A compound well signal equal to (0.5)(normalized NC) was set to a normalized activity of -50%.
A compound well signal equal to (2)(normalized NC) was set to a normalized activity of 100%.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.

The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' from Genedata Condoseo (v7.0.3) was applied.

IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.

For active compounds activity score 38-68, for inactive score is 0.

Grant Number: 1 R03 MH086465-01