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BioAssay: AID 651996

SAR analysis of small molecule inhibitors of SKN-1 in a fluorescence ratio assay

Nematodes parasitize ~1/3 of humans world-wide. Helminth targeting drugs, or anthelmintics, have been used to control parasitic nematodes for decades and many species are evolving multidrug resistance. Indiverse organisms, multidrug resistance is mediated by increased expression and activity of enzymes that detoxify xenobiotics. Pharmacological compounds that target xenobiotic detoxification more ..
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 Tested Compounds
 Tested Compounds
All(58)
 
 
Active(48)
 
 
Inactive(10)
 
 
 Tested Substances
 Tested Substances
All(58)
 
 
Active(48)
 
 
Inactive(10)
 
 
AID: 651996
Data Source: Burnham Center for Chemical Genomics (SBCCG-A946-SKN1-Inh-DryPowder-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-01-26
Hold-until Date: 2013-11-08
Modify Date: 2013-11-08

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 48
Depositor Specified Assays
AIDNameTypeComment
624304uHTS identification of SKN-1 Inhibitors in a fluoresence assayscreening
624318Summary assay for small molecule inhibitors of SKN-1summary
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: 1R21NS067678-01
Assay Provider: Keith Choe, Ph.D., University of Florida, Florida

Nematodes parasitize ~1/3 of humans world-wide. Helminth targeting drugs, or anthelmintics, have been used to control parasitic nematodes for decades and many species are evolving multidrug resistance. Indiverse organisms, multidrug resistance is mediated by increased expression and activity of enzymes that detoxify xenobiotics. Pharmacological compounds that target xenobiotic detoxification pathways would provide much needed tools for studying multidrug resistance and could greatly increase the useful life of current and future anthelmintics. Few pharmacological compounds are available for studying and targeting multidrug resistance and those that are available are not specific for nematodes and only target a single enzyme or class of enzymes.

The transcription factor SKN-1 activates the expression of ~100 genes predicted to promote drug modification, conjugation, or export (1-2). SKN-1 is also essential for the development of nematode embryos. Our recent studies have identified a major pathway regulating SKN-1 that is distinct from the major pathway regulating xenobiotic detoxification in mammals. SKN-1 also binds to target DNA by a unique monomeric mechanism relative to all other known basic leucine zipper factors (3,4). Therefore, SKN-1 is a promising target for the development of drugs that disrupt embryonic development, decrease stress resistance, and inhibit xenobiotic detoxification in nematodes without affecting homologous pathways in humans. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal system to screen for inhibitors of xenobiotic detoxification genes.

The goal of this assay is to confirm hits in "uHTS identification of SKN-1 inhibitors in a fluorescence assay." AID 624304 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

REFERENCES
1. Oliveira RP, Abate JP, Dilks K, Landis J, Ashraf J, Murphy CT, and Blackwell TK. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8:524-541, 2009.
2. Park S-K, Tedesco PM, and Johnson TE. Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1. Aging Cell 8: 258-269, 2009.
3.Blackwell TK, Bowerman B, Priess JR, and Weintraub H. Formation of a monomeric DNA binding domain by SKN-1 bZIP and homeodomain elements. Science 266: 621-628, 1994.
4.Hasegawa K, and Miwa J. Genetic and cellular characterization of Caenorhabditis elegans mutants
abnormal in the regulation of many phase II enzymes. PLoS ONE 5: e11194, 2010.
Protocol
Assay Materials:
Worm strain VP596 (Pgst-4::GFP;Pdop-3RFP) (Assay Provider)
Acrylamide SIgma Aldrich A8887)
LB LB Broth (Research Products L24066-1000)
Terrific Broth (Research Products T15100-5000)
Sealing Tape (Fisher Scientific 1256705)
Aurora Black HiBAse 1536-well plates

I. Compound Addition:
1- Transfer test compounds to columns 5-45 and DMSO to columns 1-4 and 45-48 using the Labcyte ECHO 555. Transfer volume of test compound is 80-2.5 nl of 10 mM stock and 40 nl - 5 nl from 0.3125 nM stock for 10pt dose-response curves in concentration range 100-0.195 M. Wells are backfilled with DMSO to 80 nl (1% DMSO).

II. worm Suspension
2- Dispense 5 uL/well of worm suspension (~20-25 worms/well)
3- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.

III. Reagent Addition
4- Dispense 3 uL/well of broth to columns 1-4.
5- Dispense 3 uL/well of acrylamide columns 5-48.
6- Spin down plates without lids on Vspin at 500 rpm for 1 min
7- Seal plates with breathable tape and incubate plates at 22 degrees C for 24 hrs.

IV. Reading plates:
8-Spin plates at 800 rpm for 1 min. Remove seals.
9-Read on envision for GFP/RFP ratio fluorescence.
Comment
Compounds that have IC50 <= 20 uM are confirmed as inhibitors of the reaction..

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.

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 in this assay

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in 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 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 85 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
Show more
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 the 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 a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the 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
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags 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
8% Activity at 64 uM_first_point (64μM**)% Activity at the test concentrationFloat%
9% Activity at 32 uM_first_point (32μM**)% Activity at the test concentrationFloat%
10% Activity at 16 uM_first_point (16μM**)% Activity at the test concentrationFloat%
11% Activity at 8 uM_first_point (8μM**)% Activity at the test concentrationFloat%
12% Activity at 4 uM_first_point (4μM**)% Activity at the test concentrationFloat%
13% Activity at 2 uM_first_point (2μM**)% Activity at the test concentrationFloat%
14% Activity at 1 uM_first_point (1μM**)% Activity at the test concentrationFloat%
15% Activity at 0.5 uM_first_point (0.5μM**)% Activity at the test concentrationFloat%
16% Activity at 0.25 uM_first_point (0.25μM**)% Activity at the test concentrationFloat%
17% Activity at 0.125 uM_first_point (0.125μM**)% Activity at the test concentrationFloat%
18Excluded_Points_second_pointFlags 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 64 uM_second_point (64μM**)% Activity at the test concentrationFloat%
20% Activity at 32 uM_second_point (32μM**)% Activity at the test concentrationFloat%
21% Activity at 16 uM_second_point (16μM**)% Activity at the test concentrationFloat%
22% Activity at 8 uM_second_point (8μM**)% Activity at the test concentrationFloat%
23% Activity at 4 uM_second_point (4μM**)% Activity at the test concentrationFloat%
24% Activity at 2 uM_second_point (2μM**)% Activity at the test concentrationFloat%
25% Activity at 1 uM_second_point (1μM**)% Activity at the test concentrationFloat%
26% Activity at 0.5 uM_second_point (0.5μM**)% Activity at the test concentrationFloat%
27% Activity at 0.25 uM_second_point (0.25μM**)% Activity at the test concentrationFloat%
28% Activity at 0.125 uM_second_point (0.125μM**)% Activity at the test concentrationFloat%
29Excluded_Points_third_pointFlags 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
30% Activity at 64 uM_third_point (64μM**)% Activity at the test concentrationFloat%
31% Activity at 32 uM_third_point (32μM**)% Activity at the test concentrationFloat%
32% Activity at 16 uM_third_point (16μM**)% Activity at the test concentrationFloat%
33% Activity at 8 uM_third_point (8μM**)% Activity at the test concentrationFloat%
34% Activity at 4 uM_third_point (4μM**)% Activity at the test concentrationFloat%
35% Activity at 2 uM_third_point (2μM**)% Activity at the test concentrationFloat%
36% Activity at 1 uM_third_point (1μM**)% Activity at the test concentrationFloat%
37% Activity at 0.5 uM_third_point (0.5μM**)% Activity at the test concentrationFloat%
38% Activity at 0.25 uM_third_point (0.25μM**)% Activity at the test concentrationFloat%
39% Activity at 0.125 uM_third_point (0.125μM**)% Activity at the test concentrationFloat%
40Excluded_Points_fourth_pointFlags 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
41% Activity at 64 uM_fourth_point (64μM**)% Activity at the test concentrationFloat%
42% Activity at 32 uM_fourth_point (32μM**)% Activity at the test concentrationFloat%
43% Activity at 16 uM_fourth_point (16μM**)% Activity at the test concentrationFloat%
44% Activity at 8 uM_fourth_point (8μM**)% Activity at the test concentrationFloat%
45% Activity at 4 uM_fourth_point (4μM**)% Activity at the test concentrationFloat%
46% Activity at 2 uM_fourth_point (2μM**)% Activity at the test concentrationFloat%
47% Activity at 1 uM_fourth_point (1μM**)% Activity at the test concentrationFloat%
48% Activity at 0.5 uM_fourth_point (0.5μM**)% Activity at the test concentrationFloat%
49% Activity at 0.25 uM_fourth_point (0.25μM**)% Activity at the test concentrationFloat%
50% Activity at 0.125 uM_fourth_point (0.125μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R21NS067678-01

Data Table (Concise)
Classification
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