Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: X01 MH083230-01
Assay Provider: Dr. Dmitri Rosenoff, Sanford-Burnham Medical Research Institute, San Diego CA

This assay is a counter screen for AID 1443, "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells".

The goal of this assay is to determine if compounds are cytotoxic to PPC-1 cells. The assay uses a luminescence readout via ATPlite (Perkin Elmer) and is based on the ATP content within cells. The more viable cells there are in a well the more ATP is available, and subsequently the higher the signal in the well. Compounds which are cytotoxic will cause a drop in the luminescent signal.

References:
Bodmer JL, Meier P, Tschopp J, Schneider P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 2000, 275:20632-7.

Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B, Hillan K, Totpal K, DeForge L, Schow P, Hooley J, Sherwood S, Pai R, Leung S, Khan L, Gliniak B, Bussiere J, Smith CA, Strom SS, Kelley S, Fox JA, Thomas D, Ashkenazi A. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 2001, 7:383-5.

Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003, 63:5390-400

Greil R, Anether G, Johrer K, Tinhofer I. Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools. J Leukoc Biol 2003, 74:311-30.

Smyth MJ, Takeda K, Hayakawa Y, Peschon JJ, van den Brink MR, Yagita H. Nature's TRAIL-on a path to cancer immunotherapy. Immunity 2003, 18:1-6.

Assay materials:

1) PPC-1 cells were provided by the assay provider
2) ATPLite (Perkin Elmer)

uHTS protocol:

Day 1
1) PPC-1 cells are grown in DMEM without phenol red, 10% FBS, 2mM L-glutamine, 1mM Na-pyruvate, 15ug/ml gentamycin and are harvested at 100% confluency.
2) Using a Biomek FX cells are seeded into a 1536 well plate (Corning #3727) at a concentration of 250 cells/well in 5ul. Cells are seeded in columns 3 through 48. Media is added to wells in columns 1 and 2 (Positive control).
3) Plates are spun in an Eppendorf model 5810 centrifuge @ 500 RPM for 5min.
4) Plates are placed in stacks of 6 (with a dummy plate filed on the top and bottom of each stack) and wrapped in Saran Wrap. This helps to eliminate any edge effects.
5) Incubate overnight in 37 oC 5% CO2 incubator

Day 2
1) Unwrap plates
2) Add 2.5 nl of 10mM compound to wells (columns 5-48).
3) Add 2.5 nl of 100% DMSO to wells in columns 1 through 4.
4) Plates are spun in an Eppendorf model 5810 centrifuge @ 500 RPM for 5min
5) Plates are placed in stacks of 6 (with a dummy plate filed on the top and bottom of each stack) and re-wrapped in Saran Wrap.
6) Incubate overnight in 37 oC 5% CO2 incubator

Day 3
1) Unwrap plates
2) Add 3ul of ATPlite to each well
3) Spin plate in a Velocity11 Vspin
4) Shake plate
5) After 20 min read luminescence using a Perkin Elmer Viewlux (read time= 10sec, filter=clear, gain=high, binning=2x)

Compounds that reconfirmed with an average of >=50% activity are defined as actives in this assay, thus are considered cytotoxic.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 5 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
Active compounds will have activity scores between 20 and 40. While inactive compounds will have activity scores between 0 and 20.
2) Second tier (41-80 range) is reserved for liquid resupply single concentration confirmation and or dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues

Grant Number: X01 MH083230-01