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BioAssay: AID 1487

qHTS Assay for Modulators of Lamin A Splicing

Hutchinson-Gilford Progeria Syndrome (HGPS) is a pediatric premature aging disease caused by a spontaneous mutation in the lamin A/C (LMNA) gene. The mutation activates a cryptic splice site in the LMNA pre-mRNA which results in production of a pre-lamin A protein that cannot be processed properly. The mutant protein accumulates in the nucleus and negatively affects numerous cellular functions. more ..
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 Tested Compounds
 Tested Compounds
All(197098)
 
 
Active(27)
 
 
Inactive(151437)
 
 
Inconclusive(46020)
 
 
 Tested Substances
 Tested Substances
All(198098)
 
 
Active(27)
 
 
Inactive(151734)
 
 
Inconclusive(46337)
 
 
AID: 1487
Data Source: NCGC (LMNA453)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-01-07
Modify Date: 2009-01-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 27
Related Experiments
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AIDNameTypeProbeComment
1498Confirmation Concentration-Response Assay for Modulators of Lamin A SplicingConfirmatory depositor-specified cross reference
2710Kinase Inhibition Profile Study on Inhibitors of CDC-like Kinase 4Other depositor-specified cross reference
488872qHTS for Inhibitors of DYRK1A: SummarySummary1 depositor-specified cross reference
488883qHTS for Inhibitors of DYRK1B: SummarySummary1 depositor-specified cross reference
488887qHTS for Inhibitors of CDC-like Kinase 1 and 4: SummarySummary1 depositor-specified cross reference
493173qHTS Assay for Modulators of Lamin A Splicing: SummarySummary depositor-specified cross reference
504421Kinase Inhibition Study on Inhibitors of CDC-like Kinase 4 (Reaction Biology data): SAR round 2Confirmatory depositor-specified cross reference
504424Kinase Inhibition Study on Inhibitors of Dyrk1a (Reaction Biology data)Confirmatory depositor-specified cross reference
504427Kinase Inhibition Study on Inhibitors of CDC-like Kinase 2 (Reaction Biology data)Confirmatory depositor-specified cross reference
504428Kinase Inhibition Study on Inhibitors of CDC-like Kinase 3 (Reaction Biology data)Confirmatory depositor-specified cross reference
504429Kinase Inhibition Study on Inhibitors of Dyrk1b (Reaction Biology data)Confirmatory depositor-specified cross reference
504430Kinase Inhibition Study on Inhibitors of CDC-like Kinase 1 (Reaction Biology data)Confirmatory depositor-specified cross reference
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
2705Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay)Confirmatory same project related to Summary assay
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2Confirmatory same project related to Summary assay
493206Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay): round 2 SARConfirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
2705Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay)Confirmatory same project related to Summary assay
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2Confirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory same project related to Summary assay
2705Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay)Confirmatory same project related to Summary assay
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2Confirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
Description:
NCGC Assay Overview:

Hutchinson-Gilford Progeria Syndrome (HGPS) is a pediatric premature aging disease caused by a spontaneous mutation in the lamin A/C (LMNA) gene. The mutation activates a cryptic splice site in the LMNA pre-mRNA which results in production of a pre-lamin A protein that cannot be processed properly. The mutant protein accumulates in the nucleus and negatively affects numerous cellular functions. Correction of the splicing defect in HGPS patient cells using a targeted oligonucleotide (exo11) leads to reversal of the cellular disease phenotypes. To identify small molecule modulators of aberrant LMNA splicing, a homogenous assay was constructed in HeLa cells that used a GFP containing minigene to report on correction of aberrant splicing in lamin A and a RFP (DsRed2) to report on cell viability, uniformity and nonspecific effects on the assay signal. This assay was adapted to laser-scanning microplate cytometry (Acumen, TTP Labtech) where miniaturization of the assay to a 1,536-well plate format was possible.

References:
Bowen, W. P.; Wylie, P. G. Application of Laser-Scanning Fluorescence Microplate Cytometry in High Content Screening. Assay Drug Dev Technol 2006;4:209-221.
Scaffidi P, Misteli T. Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat. Med. 2005;11:440-445.
Protocol
NCGC Assay Protocol Summary:
Reagents/Controls: Stable clones expressing DsRed2 and lamin A-EGFP were generated by Cell and Molecular Technologies in S3 HeLa cells.
HeLa cells were prepared for the assay when they were 70-90% confluent. The cells were counted and resuspended in Opti-MEM I (no phenol red; Gibco), 2% FBS, 1% penicillin/ streptomycin (Gibco) at concentration of 120 cells/uL concentration. The suspension was passed through a Falcon basket filter with pore size 40um (Beckton Dickson).
Control titrations: column of the 1536-well plate contained a sixteen point titration (1:2 dilutions) in duplicate of ancitabine (Sigma: A8598) beginning at 40 uM final concentration. Column 2 contained a sixteen point titration (1:2 dilutions) of cycloheximide beginning at 1.4 mM final concentration. Column 3 contained ancitabine at 40 uM and column 4 contained was the vehicle control (0.5% DMSO final concentration).
Assay Steps:
5 uL of HeLa cells were plated in Aurora LoBase black clear bottom (cyclic-olefin polymer) 1536-well plates at a final density of 600 cells/well. Controls and library compounds were added with a 1536-well pin array using the Kalypsys pin-tool. The plates were then incubated for 48 hrs in a cell-culture incubator (37oC; 5% CO2, 98% humidity). Following incubation the plates were read on the Acumen Explorer using the 488 nm laser. Data was collected on objects between 10 and 100 um in size. Cell objects were defined by a 15 um width x 10 um depth filter. The ratio of the total object intensity in PMT1 (500-530 nm emission; GFP) over PMT3 (575-640 nm emission; RFP) was calculated for analysis.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, lamin A, Progeria, RNA splicing.
Comment
Compound Ranking:
1. Compounds are first classified as being probable inhibitors (compounds decreasing ratio signal), activators (compounds increasing ratio signal), fluorescent (artifactual results), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description".
3. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active inhibitor 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. Ratio-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HeLa
From ChEMBL:
Assay Type: Functional
Result Definitions
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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
5RFP-Curve_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 signficant, but below 80% of control.String
6RFP-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7RFP-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8RFP-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9RFP-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10RFP-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11RFP-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12RFP-Excluded_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
13RFP-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14RFP-Activity at 0.0002635945 uM (0.000263594μM**)% Activity at given concentration.Float%
15RFP-Activity at 0.0005899385 uM (0.000589939μM**)% Activity at given concentration.Float%
16RFP-Activity at 0.00140 uM (0.00140009μM**)% Activity at given concentration.Float%
17RFP-Activity at 0.00281 uM (0.00281268μM**)% Activity at given concentration.Float%
18RFP-Activity at 0.00295 uM (0.00294944μM**)% Activity at given concentration.Float%
19RFP-Activity at 0.00565 uM (0.00564683μM**)% Activity at given concentration.Float%
20RFP-Activity at 0.011 uM (0.0112507μM**)% Activity at given concentration.Float%
21RFP-Activity at 0.015 uM (0.0147471μM**)% Activity at given concentration.Float%
22RFP-Activity at 0.023 uM (0.0227748μM**)% Activity at given concentration.Float%
23RFP-Activity at 0.045 uM (0.0447398μM**)% Activity at given concentration.Float%
24RFP-Activity at 0.074 uM (0.0737351μM**)% Activity at given concentration.Float%
25RFP-Activity at 0.090 uM (0.0900058μM**)% Activity at given concentration.Float%
26RFP-Activity at 0.179 uM (0.178791μM**)% Activity at given concentration.Float%
27RFP-Activity at 0.360 uM (0.360023μM**)% Activity at given concentration.Float%
28RFP-Activity at 0.369 uM (0.368672μM**)% Activity at given concentration.Float%
29RFP-Activity at 0.724 uM (0.72392μM**)% Activity at given concentration.Float%
30RFP-Activity at 1.440 uM (1.44009μM**)% Activity at given concentration.Float%
31RFP-Activity at 1.843 uM (1.84335μM**)% Activity at given concentration.Float%
32RFP-Activity at 2.913 uM (2.91262μM**)% Activity at given concentration.Float%
33RFP-Activity at 5.724 uM (5.72411μM**)% Activity at given concentration.Float%
34RFP-Activity at 9.217 uM (9.21666μM**)% Activity at given concentration.Float%
35RFP-Activity at 11.52 uM (11.5207μM**)% Activity at given concentration.Float%
36RFP-Activity at 22.86 uM (22.8551μM**)% Activity at given concentration.Float%
37RFP-Activity at 46.00 uM (46μM**)% Activity at given concentration.Float%
38RFP-Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
39GFP-Curve_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 signficant, but below 80% of control.String
40GFP-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
41GFP-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
42GFP-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
43GFP-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
44GFP-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
45GFP-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
46GFP-Excluded_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
47GFP-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
48GFP-Activity at 0.0002635945 uM (0.000263594μM**)% Activity at given concentration.Float%
49GFP-Activity at 0.0005899385 uM (0.000589939μM**)% Activity at given concentration.Float%
50GFP-Activity at 0.00140 uM (0.00140009μM**)% Activity at given concentration.Float%
51GFP-Activity at 0.00281 uM (0.00281268μM**)% Activity at given concentration.Float%
52GFP-Activity at 0.00295 uM (0.00294944μM**)% Activity at given concentration.Float%
53GFP-Activity at 0.00565 uM (0.00564683μM**)% Activity at given concentration.Float%
54GFP-Activity at 0.011 uM (0.0112507μM**)% Activity at given concentration.Float%
55GFP-Activity at 0.015 uM (0.0147471μM**)% Activity at given concentration.Float%
56GFP-Activity at 0.023 uM (0.0227748μM**)% Activity at given concentration.Float%
57GFP-Activity at 0.045 uM (0.0447398μM**)% Activity at given concentration.Float%
58GFP-Activity at 0.074 uM (0.0737351μM**)% Activity at given concentration.Float%
59GFP-Activity at 0.090 uM (0.0900058μM**)% Activity at given concentration.Float%
60GFP-Activity at 0.179 uM (0.178791μM**)% Activity at given concentration.Float%
61GFP-Activity at 0.360 uM (0.360023μM**)% Activity at given concentration.Float%
62GFP-Activity at 0.369 uM (0.368672μM**)% Activity at given concentration.Float%
63GFP-Activity at 0.724 uM (0.72392μM**)% Activity at given concentration.Float%
64GFP-Activity at 1.440 uM (1.44009μM**)% Activity at given concentration.Float%
65GFP-Activity at 1.843 uM (1.84335μM**)% Activity at given concentration.Float%
66GFP-Activity at 2.913 uM (2.91262μM**)% Activity at given concentration.Float%
67GFP-Activity at 5.724 uM (5.72411μM**)% Activity at given concentration.Float%
68GFP-Activity at 9.217 uM (9.21666μM**)% Activity at given concentration.Float%
69GFP-Activity at 11.52 uM (11.5207μM**)% Activity at given concentration.Float%
70GFP-Activity at 22.86 uM (22.8551μM**)% Activity at given concentration.Float%
71GFP-Activity at 46.00 uM (46μM**)% Activity at given concentration.Float%
72GFP-Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
73Ratio-Curve_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 signficant, but below 80% of control.String
74Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
75Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
76Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
77Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
78Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
79Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
80Ratio-Excluded_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
81Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
82Ratio-Activity at 0.0002635945 uM (0.000263594μM**)% Activity at given concentration.Float%
83Ratio-Activity at 0.0005899385 uM (0.000589939μM**)% Activity at given concentration.Float%
84Ratio-Activity at 0.00140 uM (0.00140009μM**)% Activity at given concentration.Float%
85Ratio-Activity at 0.00281 uM (0.00281268μM**)% Activity at given concentration.Float%
86Ratio-Activity at 0.00295 uM (0.00294944μM**)% Activity at given concentration.Float%
87Ratio-Activity at 0.00565 uM (0.00564683μM**)% Activity at given concentration.Float%
88Ratio-Activity at 0.011 uM (0.0112507μM**)% Activity at given concentration.Float%
89Ratio-Activity at 0.015 uM (0.0147471μM**)% Activity at given concentration.Float%
90Ratio-Activity at 0.023 uM (0.0227748μM**)% Activity at given concentration.Float%
91Ratio-Activity at 0.045 uM (0.0447398μM**)% Activity at given concentration.Float%
92Ratio-Activity at 0.074 uM (0.0737351μM**)% Activity at given concentration.Float%
93Ratio-Activity at 0.090 uM (0.0900058μM**)% Activity at given concentration.Float%
94Ratio-Activity at 0.179 uM (0.178791μM**)% Activity at given concentration.Float%
95Ratio-Activity at 0.360 uM (0.360023μM**)% Activity at given concentration.Float%
96Ratio-Activity at 0.369 uM (0.368672μM**)% Activity at given concentration.Float%
97Ratio-Activity at 0.724 uM (0.72392μM**)% Activity at given concentration.Float%
98Ratio-Activity at 1.440 uM (1.44009μM**)% Activity at given concentration.Float%
99Ratio-Activity at 1.843 uM (1.84335μM**)% Activity at given concentration.Float%
100Ratio-Activity at 2.913 uM (2.91262μM**)% Activity at given concentration.Float%
101Ratio-Activity at 5.724 uM (5.72411μM**)% Activity at given concentration.Float%
102Ratio-Activity at 9.217 uM (9.21666μM**)% Activity at given concentration.Float%
103Ratio-Activity at 11.52 uM (11.5207μM**)% Activity at given concentration.Float%
104Ratio-Activity at 22.86 uM (22.8551μM**)% Activity at given concentration.Float%
105Ratio-Activity at 46.00 uM (46μM**)% Activity at given concentration.Float%
106Ratio-Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
107Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084827-01

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