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BioAssay: AID 493175

miR-21 counterscreen using purified firefly luciferase

MicroRNAs (miRNAs) are endogenously encoded small (~20-25 nucleotides), nonprotein-coding RNAs that are involved in post-transcriptional repression of target messenger RNAs (mRNAs) (Le and Hannon, 2004). Estimated to be involved in the regulation of 30% of all protein-coding mRNAs (Filipowicz et al., 2008), miRNAs play a significant role in many biological processes including cellular more ..
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 Tested Compounds
 Tested Compounds
All(124)
 
 
Inactive(119)
 
 
Inconclusive(5)
 
 
 Tested Substances
 Tested Substances
All(124)
 
 
Inactive(119)
 
 
Inconclusive(5)
 
 
AID: 493175
Data Source: NCGC (MIRNA004)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-02-09
Modify Date: 2011-02-23

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
2288qHTS Assay for Modulators of miRNAs and/orActivators of miR-21confirmatoryqHTS assay 1
2289qHTS Assay for Modulators of miRNAs and/or Inhibitors of miR-21confirmatoryqHTS assay 2
2507Secondary Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21: miR-30 Firefly Luciferase Assayconfirmatoryconfirmation miR-30 assay
2508Confirmatory Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21confirmatoryconfirmation miR-21 assay
2598qHTS Assay for General miRNAs Modulators and/or Inhibitors/Activators of miR-21: Summarysummarysummary assay
Description:
MLPCN Grant: 1 R21 NS059478-01

Assay Provider: Qihong Huang, The Wistar Institute

NCGC Assay Overview:

MicroRNAs (miRNAs) are endogenously encoded small (~20-25 nucleotides), nonprotein-coding RNAs that are involved in post-transcriptional repression of target messenger RNAs (mRNAs) (Le and Hannon, 2004). Estimated to be involved in the regulation of 30% of all protein-coding mRNAs (Filipowicz et al., 2008), miRNAs play a significant role in many biological processes including cellular differentiation and tissue development (Ghildiyal and Zamore, 2009). Not surprisingly, miRNAs have been found to be oncogenic or tumor suppressive, with altered miRNA expression levels associated with human cancers (Esquela-Kerscher and Slack, 2006). The miRNA miR-21 has been found to be expressed in most human tissues (Sonkoly et al., 2007), and its overexpression has been associated with a variety of human tumors (Tong and Nemunanitis, 2008). miR-21 has been shown to be anti-apoptotic: knock-down of miR-21 in a glioblastoma cell line was found to activate caspases and lead to apoptosis (Chan et al., 2005). Identification of small molecules that modulate miR-21 specifically, or miRNAs generally, would prove useful as tools to better understand miRNA biogenesis and mRNA regulation by miRNAs. Of special interest are compounds that specifically inhibit miR-21, and a cell-based firefly luciferase reporter gene assay optimized for 1536-well format quantitative HTS (qHTS) was developed and performed in an effort to identify such compounds.

Compounds active in the miR-21 FLuc assay were clustered using Leadscope and active chemical series were prioritized based on chemical properties, potential for medicinal chemistry optimization, and SAR. Within each chemical series, representative compounds were re-acquired to test for activity in a secondary assay based on the quality of their CRC, efficacy, and potency. This secondary assay to determine if compounds active in the miR-21 FLuc assay were just luciferase inhibitors. Compounds found active in both the miR-21 FLuc and purified FLuc assays are considered as false positives.

Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, firefly luciferase, FLuc, miRNA.
Protocol
NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary: Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60muM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007360000 uM (0.000736μM**)% Activity at given concentration.Float%
15Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
16Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
17Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
18Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
19Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
20Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
21Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R21 NS059478-01

Data Table (Concise)
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