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 ..
BioActive Compounds: 48
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.
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.
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.
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 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)