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BioAssay: AID 2800

SAR analysis of compounds that are cytotoxic to HEK293 - Set 3

The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis. ..more
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AID: 2800
Data Source: Burnham Center for Chemical Genomics (BCCG-A347-HEK-293-DryPowder-Assay-3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-04-29
Hold-until Date: 2010-09-14
Modify Date: 2011-01-06

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
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AIDNameTypeProbeComment
1575Summary assay for the identification of compounds that inhibit NOD1summary2 Summary AID.
1578uHTS luminescence assay for the identification of compounds that inhibit NOD1confirmatory
1848uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD2 cell inhibition assayconfirmatory
1849uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assayconfirmatory
2001uHTS luminescence assay for the identification of compounds that inhibit NOD2 in MDP treated cellsconfirmatory
2333SAR analysis of compounds that inhibit NOD1 revisedconfirmatory
2334SAR analysis of compounds that inhibit NOD2 revisedconfirmatory
2335SAR analysis of compounds that are cytotoxic to HEK293 revisedconfirmatory
2337SAR analysis of inhibitors of TNFa specific NF-kB induction revisedconfirmatory
2469SAR analysis of compounds that are cytotoxic to HEK293 - Set 2confirmatory
Description:
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: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA

The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.

Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn's disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-kB activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models.

This dose response assay is developed and performed to confirm hits originally identified in "uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assay" AID 1849 and "uHTS Fluorescence assay for the identification of cytotoxic compounds" among compounds active in NOD2 cell inhibition assay 1848 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

Resazurin detects cell viability by converting from a nonfluorescent dye to the red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth. Continued cell growth maintains a reduced environment while inhibition of growth maintains an oxidized environment. Reduction related to growth causes the REDOX indicator to change from the oxidized (nonfluorescent, purple color) form to the reduced (fluorescent, red color) form. The fluorescent signal is monitored using 531 nm excitation wavelength and 595 nm emission wavelength.

This assay could be multiplexed with one or more of the following assays:
1) NOD1, the protocol is described in AID 2333.
2) NOD2, the protocol is described in AID 2334.
3) TNFa, the protocol is described in AID 2337.
Protocol
Protocol:
Assay materials:
1) HEK-293-T NF-kB-Luc cell line obtained from the assay provider's laboratory.
2) gamma-tri-DAP (Ana Spec cat #60774) obtained from assay provider's laboratory.
3) Resazurin (Sigma cat# R7017-5G)

Dose response cytotoxity assay for HEK-293-T NF-kB-Luc cells

Day 1 Procedure

1) Harvest HEK-293-T NFKB-Luc at 100% confluency at 100% confluency
2) Add 1 uL/well NOD assay media with Multidrop
3) Spin down plates at 1000 rpm for 1 min in an Eppendorf 5810 centrifuge.
4) Serial compound dilutions: dispense with Labcyte Echo 550 50 nl total volume 100% DMSO (DPI compounds columns 5-46 & DMSO controls col 1-4, 47-48) to plates from step 2.
5) Add gamma-tri-DAP to cell suspension at 0.75 ug/mL.
6) Seed 13000 cells/well in 4 uL/well to full plate HEK-293-T NFKB-Luc to Corning # 3727 white, 1536, hi-profile, TC-treated plate.
7) Spin down plates @ 500 RPM for 5 min on Eppendorf 5810 centrifuge.
8) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with H2O
9) Wrap plates securely in single layer of Plastic Wrap (Saran Wrap PVDC version).
10) Incubate overnight (14 hours) in 37 oC 5% CO2 incubator

Day 2 Procedure
1) Add 20 nL Resazurin 2.2 mM Resarurin in 50%/50% DMSO/dH20 using HighRes biosolution pintool equipped with V&P Scientific pins.
2) Wrap and incubate plates 1 hr in 37 oC 5% CO2 incubator
3) Top read Fluorescence intensity on Envision. (Bodipy dichroic, 595nM emission, 531nM excitation filter).
Comment
Compounds were tested in 1 or more ranges.

Range1 0-20 uM

Compounds are considered active if the IC50_Mean < 20 uM.

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 and is not applicable to this assay

2) 2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues

a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3Excluded_Points_first_point_Range1Flags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
4IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
5IC50_1_Range1IC50 value determined using sigmoidal dose response equationFloatμM
6Std.Err(IC50)_1_Range1Standard Error of IC50 valueFloatμM
7nH_1_Range1Hill coefficient determined using sigmoidal dose response equationFloat
8% Activity at 20 uM_first_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
9% Activity at 10 uM_first_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
10% Activity at 5 uM_first_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
11% Activity at 2.5 uM_first_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
12% Activity at 1.25 uM_first_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
13% Activity at 0.625 uM_first_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
14% Activity at 0.3125 uM_first_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
15% Activity at 0.15625 uM_first_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
16% Activity at 0.078125 uM_first_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
17% Activity at 0.0390625 uM_first_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%
18Excluded_Points_second_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 20 uM_second_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
20% Activity at 10 uM_second_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
21% Activity at 5 uM_second_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
22% Activity at 2.5 uM_second_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
23% Activity at 1.25 uM_second_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
24% Activity at 0.625 uM_second_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
25% Activity at 0.3125 uM_second_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
26% Activity at 0.15625 uM_second_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
27% Activity at 0.078125 uM_second_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
28% Activity at 0.0390625 uM_second_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%
29IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, IC50_2. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2_Range1IC50 value determined using sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2_Range1Standard Error of IC50 valueFloatμM
32nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloatμM
33Excluded_Points_third_point_Range1Flags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 20 uM_third_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
35% Activity at 10 uM_third_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
36% Activity at 5 uM_third_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
37% Activity at 2.5 uM_third_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
38% Activity at 1.25 uM_third_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
39% Activity at 0.625 uM_third_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
40% Activity at 0.3125 uM_third_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
41% Activity at 0.15625 uM_third_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
42% Activity at 0.078125 uM_third_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
43% Activity at 0.0390625 uM_third_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%
44Excluded_Points_fourth_point_Range1Flags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 20 uM_fourth_point_Range1 (20μM**)% Inhibition at a given concentrationFloat%
46% Activity at 10 uM_fourth_point_Range1 (10μM**)% Inhibition at a given concentrationFloat%
47% Activity at 5 uM_fourth_point_Range1 (5μM**)% Inhibition at a given concentrationFloat%
48% Activity at 2.5 uM_fourth_point_Range1 (2.5μM**)% Inhibition at a given concentrationFloat%
49% Activity at 1.25 uM_fourth_point_Range1 (1.25μM**)% Inhibition at a given concentrationFloat%
50% Activity at 0.625 uM_fourth_point_Range1 (0.625μM**)% Inhibition at a given concentrationFloat%
51% Activity at 0.3125 uM_fourth_point_Range1 (0.3125μM**)% Inhibition at a given concentrationFloat%
52% Activity at 0.15625 uM_fourth_point_Range1 (0.15625μM**)% Inhibition at a given concentrationFloat%
53% Activity at 0.078125 uM_fourth_point_Range1 (0.078125μM**)% Inhibition at a given concentrationFloat%
54% Activity at 0.0390625 uM_fourth_point_Range1 (0.0390625μM**)% Inhibition at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084844-01

Data Table (Concise)
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