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BioAssay: AID 1740

Confirmation Concentration-Response Assay for Enhancers of SMN2 Splice Variant Expression

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN called SMN1 and SMN2 which are 99% identical at the amino acid level. SMN1 is a fully functional protein and SMN2 skips exon 7 90% of the time. Skipping of exon 7 produces non-functional protein product. 10% of the SMN2 more ..
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 Tested Compounds
 Tested Compounds
All(84)
 
 
Active(8)
 
 
Inactive(63)
 
 
Inconclusive(8)
 
 
Unspecified(5)
 
 
 Tested Substances
 Tested Substances
All(88)
 
 
Active(8)
 
 
Inactive(63)
 
 
Inconclusive(12)
 
 
Unspecified(5)
 
 
AID: 1740
Data Source: NCGC (SMN2004)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-05-08

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 8
Depositor Specified Assays
AIDNameTypeProbeComment
1458qHTS Assay for Enhancers of SMN2 Splice Variant Expressionconfirmatory
1474Quantitative High-Throughput Screen for Enhancers of SMN2 Splice Variant Expression: Summarysummary2
2514Confirmation Concentration-Response Assay for Enhancers of SMN2 Splice Variant Expression for Probe SARconfirmatory
2513Counterscreen Assay for Enhancers of SMN2 Splice Variant Expression: Modulation of SMN1 Expression for Probe SARconfirmatory
2515Counterscreen Assay for Enhancers of SMN2 Splice Variant Expression: Interaction with Luciferase Reporter for Probe SARconfirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: R03 MH084179-01
Assay Submitter (PI): Elliot Androphy

NCGC Assay Overview:

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN called SMN1 and SMN2 which are 99% identical at the amino acid level. SMN1 is a fully functional protein and SMN2 skips exon 7 90% of the time. Skipping of exon 7 produces non-functional protein product. 10% of the SMN2 protein includes exon 7 and is fully functional. In the SMA disease state, mutations in the SMN1 locus are the cause of the disease state. Because only 10% of SMN2 is of the fully functional form, it is not sufficient to overcome the deficiency produced by the loss of the SMN1 product. A therapy that either increase the amount of SMN2 product made or to increase the inclusion of exon 7 has been proposed for the treatment of SMA.

We have designed an assay to identify small molecules that can increase the amount of functional SMN2 product by appending a luciferase reporter gene after the native SMN2 gene, such that inclusion of exon 7 in the expressed product places the luciferase sequence in frame, thus generating functional luciferase enzyme.
Protocol
NCGC Assay Protocol Summary:

This screen utilizes a luciferase reporter gene assay, combining the promoter with the major portion of the native SMN2 cDNA (exons 1 -7, intron 8 , exon 8 fused to luciferase) stably expressed in HEK293 cells. Compounds that increase luciferase signal presumably enhance expression of the functional SMN2 splice variant.

Passaging media contained DMEM w/ glutamax (+phenol red) 10% FCS, 1x pen/strep, 200 ug/ml hygro, 1x sodium pyruvate.
Assay media contained DMEM w/ glutamax (-phenol red) 10% FCS, 1x pen strep, 1x pyruvate

Sequence, Parameter, Value, Description
(1) Cells, 5 uL, 2000 cells/well, 1536 TC treated White solid bottom plate
(2) Time, 10-12 hours, 37C 5% CO2
(3) Compound, 23 nl, MLSMR Library
(4) Control Compound, 23 nL, Sodium butyrate st 4.5mM (final) conc
(5) Time, 30-36 hours, 37C 5% CO2
(6) Reagent, 3 ul, One Glo (TM) from Promega
(7) Time, 5-15 minutes, Room temp
(8) Detector, Viewlux: luminescent read, 60 second integration, high speed 2x binning

Keywords: Spinal muscular atrophy, SMA, SMN2, high throughput screening, MLSMR, MLPCN, NIH Roadmap, qHTS and NCGC
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 0.0000054935 uM (5.49352e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000109870 uM (1.0987e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000219741 uM (2.19741e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000439481 uM (4.39481e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0001059811 uM (0.000105981μM**)% Activity at given concentration.Float%
19Activity at 0.0001757925 uM (0.000175793μM**)% Activity at given concentration.Float%
20Activity at 0.0003647283 uM (0.000364728μM**)% Activity at given concentration.Float%
21Activity at 0.0007082235 uM (0.000708223μM**)% Activity at given concentration.Float%
22Activity at 0.00129 uM (0.00128698μM**)% Activity at given concentration.Float%
23Activity at 0.00278 uM (0.00278003μM**)% Activity at given concentration.Float%
24Activity at 0.00449 uM (0.0044897μM**)% Activity at given concentration.Float%
25Activity at 0.011 uM (0.0107208μM**)% Activity at given concentration.Float%
26Activity at 0.021 uM (0.0210713μM**)% Activity at given concentration.Float%
27Activity at 0.026 uM (0.0264828μM**)% Activity at given concentration.Float%
28Activity at 0.046 uM (0.0460658μM**)% Activity at given concentration.Float%
29Activity at 0.092 uM (0.0922458μM**)% Activity at given concentration.Float%
30Activity at 0.181 uM (0.181126μM**)% Activity at given concentration.Float%
31Activity at 0.322 uM (0.321635μM**)% Activity at given concentration.Float%
32Activity at 0.772 uM (0.771896μM**)% Activity at given concentration.Float%
33Activity at 1.457 uM (1.457μM**)% Activity at given concentration.Float%
34Activity at 2.720 uM (2.7203μM**)% Activity at given concentration.Float%
35Activity at 5.714 uM (5.71394μM**)% Activity at given concentration.Float%
36Activity at 9.475 uM (9.47544μM**)% Activity at given concentration.Float%
37Activity at 22.68 uM (22.6772μM**)% Activity at given concentration.Float%
38Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
39Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084179-01

Data Table (Concise)
Classification
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