AID	Name	Type	Comment
435007	Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition Project	summary	Summary assay

Keywords: Type I diabetes, INS-1E cells, Caspase, cytokine, apoptosis

Assay Overview:

The goal of this project is to identify probes that inhibit apoptosis induced by cytokines in the rat insulinoma cell line, INS-1E. The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as Caspase 3. INS1-E rat insulinoma cells are treated with 50 ng/mL interferon-gamma, 25 ng/mL Tumor necrosis factor-alpha and 10 ng/mL Interleukin-1-beta for 48 hours in the presence of 100 nL compounds that were leads from the primary screen. A range of concentrations of compound are used from 0.2 uM to 25 uM and IC50 values will be determined. Cells are assayed with Promega's Caspase Glo 3/7, a luciferase-based reagent for measurement of caspase activity.

Expected Outcome:
A compound that prevents the induction of or reduces Caspase-3 activity will decrease the luminescence signal in a dose-dependent manner. These are the type of compounds that will be interesting for further studies. Some compounds will not prevent Caspase activation and will no longer be considered for the project. Compounds should have an IC50 of less than 10 uM.

The cytokines induce apoptosis in primary islet beta cells and in the model cell line, INS-1E cells. The primary assay measures cellular viability but not activity of any components of the apoptosis pathway. This assay measures the levels of Caspase 3 and 7 activity in cells with a luminescence-based assay from Promega called Caspase Glo 3/7. It is known that cytokine treatment leads to a 3 to 4 fold increase in Caspase activity after 48 hours of treatment. INS-1E cells will be treated with cytokines and various concentrations of compounds for 48 hours. The compounds that decrease Caspase activity will be considered for additional studies. In addition, any compound that increases the luminescence signal due to structural properties will be eliminated because these tend to increase the level of luminescence in a dose-dependent manner. The positive control is suberoylanilide hydroxamic acid (SAHA).

Protocol:
Day 0: Collect cells and seed 8,000 cells/well of INS-1E rat beta-cell line in 30 uL using white-walled, bar coded, 384-well plates Corning 8867; incubate at 37 deg C overnight
Day 1: Add 10 uL medium with cytokine cocktail to each well using the Combi (Cocktail is 250 mL INS1E media, 92.5 uL IFN-gamma, 32.5 uL TNF-alpha and 20 uL IL-1-beta).
Day 3: Add 20 uL Caspase-Glo 3/7 reagent to each well.
Agitate with 'Big Bear' for 15 seconds to maximize cell lysis. Incubate 1 hour at room temperature.
Use Perkin Elmer Envision to read plate luminescence with ultra sensitive settings.

Cell media:
RPMI 1640 (Phenol red free), 5% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ug/ml streptomycin
Cytokines: 10 ng/mL IL-1 beta (R&D Systems, 501-RL-050), 25 ng/mL TNF-alpha (R&D Systems, 410-MT-050), 50 ng/mL IFN-gamma (Sigma, I4777-.1MG)

PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate.


EXPECTED OUTCOME: Active compounds result in decreasing readout signal.


ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).


NORMALIZATION:
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 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).
Experimental wells values were scaled to this range.


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.


MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.


PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null

Activity_Outcome = 2 (active) when:
AC <= AC_limit

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


PUBCHEM_ACTIVITY_SCORE:

PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)

PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:

120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.


Note:

The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.

All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

Grant Number: DP2 DK083048