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BioAssay: AID 1817

uHTS identification of small molecule antagonists of the binding of Siah-1 and a peptide ligand via a fluorescence polarization assay.

Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of more ..
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 Tested Compounds
 Tested Compounds
All(292329)
 
 
Active(50)
 
 
Inactive(292279)
 
 
 Tested Substances
 Tested Substances
All(292483)
 
 
Active(50)
 
 
Inactive(292433)
 
 
AID: 1817
Data Source: Burnham Center for Chemical Genomics (BCCG-A208-Siah-Antagonist-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-16
Modify Date: 2011-01-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 50
Related Experiments
AIDNameTypeComment
1826Summary small molecule antagonists of the binding of Siah-1 and a peptide ligand via a fluorescence polarization assay.Summarydepositor-specified cross reference
2127HTS fluorescence polarization-based dose response confirmatory screen for the Siah-1 primary assay utilizing an alternative fluorophore, fluorescein-labeled plectinConfirmatorydepositor-specified cross reference
2141HTS TR-FRET-based dose response confirmatory assay for Siah-1Confirmatorydepositor-specified cross reference
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 MH086475-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego CA

Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of highly conserved RING domain proteins, which regulate a variety of cellular functions, including cell cycle arrest, tumor suppression, and apoptosis through the beta-catenin degradation pathway. Siah-1 has also been identified as a p53-inducible gene, functionally linking it to an important tumor suppressor. Chemical modulators of the Siah-1 pathway would provide powerful research tools for elucidating the roles of this signaling pathway in cancer development and progression.

This work employs the use of a uHTS assay based upon fluorescence polarization, and utilizing a peptide ligand of Siah-family proteins with an attached flurophore.
Protocol
Assay materials:
1) Rhodamine-Plectin and Siah provided by the assay provider
2) Assay buffer: 25mM Bis-Tris pH 7.0, 1mM TCEP and 0.005% Tween 20
3) Siah/Rd-Plectin Assay mix: 0.005uM Rd-Plectin, 0.016uM SIAH in Assay Buffer
uHTS Protocol
1) Add 6 uL of Rd-Plectin (Positive Control) to Columns 1-2 of a black, opaque, Corning 1536-well plate (Cat#3724) using a Multidrop Combi.
2) Add 6 uL of Siah/Rd Plectin Assay Mix to Columns 3-48 using a Multidrop Combi.
3) Using a HighResolution Biosolutions pintool dispense 60nl of:
100% DMSO to columns 1-4 (1% final)
2mM Compounds in 100% DMSO to columns 5-48 (20uM final)
4) Following a 1 hr incubation at room temperature, in the dark, the plates are read on a BMG Pherastar using Rhodamine FP filters.
Dose Response Protocol
1) Add 6 uL of Rd-Plectin (Positive Control) to Columns 1-2 of a black, opaque, Corning 1536-well plate (Cat#3724) using a Multidrop Combi.
2) Add 6 uL of Siah/Rd Plectin Assay Mix to Columns 3-48 using a Multidrop Combi.
3) Using a Labcyte Echo, DMSO and test compounds are transferred to wells. DMSO only is transferred to columns 1-3 and 46-48(Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations. A total of 60 nL is added to each well of the assay plate.
4) Following a 1 hr incubation at room temperature, in the dark, the plates are read on a BMG Pherastar using Rhodamine FP filters.
Comment
Compounds with greater than or equal to 50% displacement of Rhodamine-Plectin in the Siah assay at 20 uM concentration and have an F_ratio parameter between 0.3 and 1.5 are defined as actives of the primary screening.
Dose response testing of 152 substances yielded 22 compounds with IC50 < 20 uM and were considered "active."
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 % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % displacement is less than 0%, then the assigned score is 0
b. If primary % displacement is greater than 100%, then the assigned score is 40
c. If primary % displacement is between 0% and 100%, then the calculated score is (%displacement)*0.4
d. If the F_ratio is < 0.3 or >1.50 then the score is set to 5.
e. Compounds with no fluorescence interference and >50% displacement have the score >= 20.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a.Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b.The score is linearly correlated with a compound's 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- or pathway-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 the Siah 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 all the items discussed above is
Score = 44 + 6*(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.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is applicable in this assay.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. 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*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloat
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%displacement at 20 uM (20μM**)% displacement in primary screeningFloat%
6F_Ratio (20μM**)Fluorescence intensity normalized to the average fluorescence intensity value of the plate negative controlsFloat%
7Mean HighMean fluorescence polarization of negative controls in the corresponding plateFloatmP
8STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatmP
9Mean LowMean fluorescence polarization of positive controls in the corresponding plateFloatmP
10STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatmP

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH086475-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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