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BioAssay: AID 925

qHTS Assay for Modulators of Hemoglobin Beta Chain Splicing

Abnormal splicing is associated with a number of diseases, including cancer and genetic diseases such as cystic fibrosis, muscular dystrophy and beta-thalassemia. Alteration of disease associated splicing patterns provides a promising target for treatment. Work in the Kole laboratory has demonstrated that modulation of aberrant and alternative splicing with splice switching oligonucleotides (SSO) leads to clinically relevant results. Mutations in the second intron of the beta-globin gene at positions are clinically relevant and cause mis-splicing which results in an out of frame gene product. ..more
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 Tested Compounds
 Tested Compounds
All(64405)
 
 
Active(39)
 
 
Inactive(64147)
 
 
Inconclusive(222)
 
 
 Tested Substances
 Tested Substances
All(65077)
 
 
Active(41)
 
 
Inactive(64810)
 
 
Inconclusive(226)
 
 
AID: 925
Data Source: NCGC (THAL594)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-20
Modify Date: 2010-07-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 39
Depositor Specified Assays
AIDNameTypeComment
1405Confirmation and Secondary Assays for Modulators of Hemoglobin Beta Chain Splicing at IVS2 654 and 705 lociconfirmatory
493171qHTS Assays for Modulators of Hemoglobin Beta Chain Splicing: Summarysummary
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080684-01
PI Name: Dr. Ryszard Kole

NCGC Assay Overview:

Modulation of Alternative Splicing as Therapy

Abnormal splicing is associated with a number of diseases, including cancer and genetic diseases such as cystic fibrosis, muscular dystrophy and beta-thalassemia. Alteration of disease associated splicing patterns provides a promising target for treatment. Work in the Kole laboratory has demonstrated that modulation of aberrant and alternative splicing with splice switching oligonucleotides (SSO) leads to clinically relevant results. Mutations in the second intron of the beta-globin gene at positions are clinically relevant and cause mis-splicing which results in an out of frame gene product.
SSOs which are antisense to the mutation site in the transcript have been shown to effectively block mis-splicing and restore proper splicing and generation of full length protein in cell lines and mouse models harboring mutations in intron 2 of b-globin.


EGFP-654 and 705 HeLa Cells as screen system.

HeLa cell lines were stably transfected with a construct in which the EGFP (enhanced green fluorescent protein) cDNA was interrupted by the second intron of the beta-globin gene (IVS2-654). The ISV2 intron contains mutations which cause mis-splicing of EGFP and premature termination of EGFP which results in a nonfluorescent protein product. Restoration of proper splicing by SSOs resulted in production of full length EGFP which generated a signal that was easily detectable by fluorescent microscopy, RT-PCR or FACS analysis. Expression of the transgene was driven by the CMV promoter. We concluded that a low molecular weight compound, which restored correct splicing of intron 2 would also generate functional EGFP protein, resulting in a fluorescent signal. Thus, the HeLa cells that express the EGFP-654 construct can be used for high throughput screening of a chemical library.
Protocol
NCGC Assay Protocol Summary:

High Throughput Screening. Freshly thawed HeLa EGFP-654 cells were seeded in 5 ul of optimem media (Invitrogen) containing 2 % fetal calf serum (Hyclone) and 1x penicillin/streptomycin (Invitrogen) at a concentration of 500 cells per well in 1536-well plates. Cells were incubated overnight at 37C in 5% CO2. The next day, cells were treated with DMSO, 500 nM antisense ISV2 654 PNA control oligo, or library compound and allowed to grow at 37C in 5% CO2 for 2 days. The PNA oligo blocked the mutation site in the intron and allowed proper (wild type) splicing to occur, resulting in full length EGFP expression and also allowed for optimization of the assay parameters. Plates were read in the Acumen Explorer (TTP Labtech) using 488 nm laser excitation and 500-520 nm emission PMT filter in a 1 x 8 uM capture mode. Fluorescnet Fluorescent objects ranging in size from 5 to 250 uM were measured and the total peak GFP intensity for each well was used for data analysis.

Keywords: Thalassemia, beta-thalassemia, beta-globin, EGFP, ISV2 654, ISV2 705, SSO, PNA, antisense, splicing, MLSMR, MLSCN, NIH Roadmap, qHTS, 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 activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds increasing GFP signal (activators), 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.

Anticipated false positives for this assay include compounds fluorescent at 500nm.
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
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 signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on molar units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio 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 0.0006000000 uM (0.0006μM**)% Activity at given concentration.Float%
15Activity at 0.00130 uM (0.0013μM**)% Activity at given concentration.Float%
16Activity at 0.00300 uM (0.003μM**)% Activity at given concentration.Float%
17Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
18Activity at 0.00660 uM (0.0066μM**)% Activity at given concentration.Float%
19Activity at 0.015 uM (0.0148μM**)% Activity at given concentration.Float%
20Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
21Activity at 0.033 uM (0.033μM**)% Activity at given concentration.Float%
22Activity at 0.074 uM (0.0738μM**)% Activity at given concentration.Float%
23Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
24Activity at 0.165 uM (0.1649μM**)% Activity at given concentration.Float%
25Activity at 0.369 uM (0.3687μM**)% Activity at given concentration.Float%
26Activity at 0.460 uM (0.4599μM**)% Activity at given concentration.Float%
27Activity at 0.825 uM (0.8245μM**)% Activity at given concentration.Float%
28Activity at 1.844 uM (1.8435μM**)% Activity at given concentration.Float%
29Activity at 2.299 uM (2.2991μM**)% Activity at given concentration.Float%
30Activity at 4.122 uM (4.1222μM**)% Activity at given concentration.Float%
31Activity at 9.217 uM (9.2172μM**)% Activity at given concentration.Float%
32Activity at 11.50 uM (11.495μM**)% Activity at given concentration.Float%
33Activity at 20.61 uM (20.6095μM**)% Activity at given concentration.Float%
34Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
35Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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