AID	Name	Type	Comment
2732	HTS for small molecule inhibitors of CHOP to regulate the unfolded protein response to ER stress	screening
493217	Summary AID for the MLPCN Project HTS for small molecule inhibitors of CHOP to regulate the unfolded protein response to ER stress	summary
652063			On Hold
652064			On Hold

NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
Assay provider: Dr. Randal Kaufman, University of Michigan
MLSCN Grant: R03MH084182-01, U54HG003918

Assay Overview

Many genetic and environmental diseases result from defective protein folding within the secretory pathway so that aberrantly folded proteins are recognized by the cellular surveillance system and retained within the endoplasmic reticulum (ER). Under conditions of malfolded protein accumulation, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins, and if unsuccessful, initiates a cell death response. Preliminary studies have shown that CHOP is a crucial factor in the apoptotic arm of the UPR; XBP1 activates genes encoding ER protein chaperones and thereby mediates the adaptive UPR response to increase clearance of malfolded proteins. Inhibition of CHOP is hypothesized to enhance survival by preventing UPR programmed cell death. There are currently no known small molecule CHOP inhibitors either for laboratory or clinical use.

To identify small molecule inhibitors of the UPR pathway, mediated by CHOP, a cell-based luciferase reporter assay using stably transfected CHO-K1 cells with luciferase driven by the CHOP promoter has been developed. The assay have been optimized and validated in 384-well format and used to screen for inhibitors of tunicamycin-induced CHOP in HTS. These identified compounds will have potential therapeutic application to diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia, lysosomal storage diseases, and alpha-1 antitrypsin deficiency.

A counterscreen assay using stable transfected CHO-XBP1 cells were developed and used as a counterscreen assay for the evaluation of positives from primary HTS of CHOP inhibitor.

The top 2091 positive compound identified from HTS of small molecule inhibitors of CHOP to regulate the unfolded protein response to ER stress (AID 2732), were tested in the confirmatory assay (triplicates at 10 uM in the primary assay). The 940 positives (>70% inhibition) from the confirmatory assay were further tested in dose-response using counterscreen assay (CHO-XBP1 cells). The dose-response tests were carried out at 7 concentrations and each concentration was tested in triplicates.

Cell lines & Reagents:
1. Cell line for counter screen assay (dose-response):
CHO cells are transfected with a reporter that contains the 26bp intron from XBP1 placed into the 5' end of luciferase (provided by assay PI). Luciferase is not translated unless the intron is removed via IRE1=mediated splicing upon activation of the UPR.
2. Cell growth media (Ham's F12 + Glutamax, 10% FBS, 1X non-essential amino acids, and penicillin/streptomycin) (Invitrogen)
3. Tunicamycin (Calbiochem)
4. SteadyGlo reagent (Promega)

Protocol:
1. 40 ul of medium containing CHO-XBP1 cells (3000-4000) were dispensed to 384 well white opaque plates (Corning # 3570) using a Multidrop combi (Thermo-Fisher Scientific). Plates were then incubated for 24 hrs at 37 degrees C, 5% CO2.
2. 0.5 ul of selected compounds (1 mM in DMSO) was added to wells using Sciclone (Caliper LifeSciences). The final concentration of compound is 10 uM for confirmatory assay. The dose-response tests were carried out at final compound concentrations at 0.16, 0.32, 0.63, 1.25, 2.5, 5 and 10 uM.
3. 10 ul of fresh medium containing tunicamycin (Tm) (2.0 ug/ml, final concentration,) was then added and the plates were incubated for 15-18 hrs.
4. Medium was aspirated with an Elx405 plate washer (BioTek), leaving 10 ul of medium in the well. 10 ul of Steady-Glo was added to each well using a multildrop combi.
5. Luminescence signal was measured on an Envision Multilable plate reader (PerkinElmer).

Data analysis:



1. Assay results are analyzed using CambridgeSoft. Percentage of inhibition is calculated with the following equation based on normalized data from each plate:

% of Control = (Luminescence compound - Luminescence blank) / (Luminescence control - Luminescence blank) * 100

Where Luminescence compound is the luminescence signal from a well with a test compound and Tm, Luminescence blank is an average luminescence signal from wells without Tm. Luminescence control is an average of luminescence signals from wells containing Tm with vehicle only.

2. Positives from confirmatory assay were further tested in dose-response counterscreening assay using CHO-XBP1 cells. The % of Control was calculated and used for IC50 analysis.

A 4 parameter logistic equation is fit to each compound using software from the R project for statistical analysis:

Y = ((Top-Bottom)/(1+(conc/X50)^Curvature))+Bottom

All of the fitted parameters are reported, as well as the absolute IC50 values (the concentration with % of control at 50)

Compounds with an IC50 < 5 were assigned a PUBCHEM_ACTIVITY_OUTCOME of Active, and all others were assigned Inactive.

PUBCHEM_ACTIVITY_SCORE is calculated by normalizing the observed range of IC50s with the lowest IC50 corresponding to 100 using the following formula:

Activity Score = (minimum IC50 value / IC50 value) * 100
100

1. Artifacts of this assay could be resulted from, but are not limited to, intrinsic fluorescence of some compounds, compounds that can quench luminescence, dust or lint and toxic compounds.

2. All data reported were normalized on a per-plate basis.

Grant Number: R03MH084182-01