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BioAssay: AID 2675

qHTS Assay for Inhibitors of MBNL1-poly(CUG) RNA binding

Expanded CTG repeats in the 3' untranslated region of the DM protein kinase (DMPK) gene causes type I myotonic dystrophy (DM1). DM1 is a multi-systemic disease with a prevalence of 1/7000. Clinical features include myotonia, progressive muscle weakness and wasting, cardiac arrhythmia, insulin resistance, hypersomnolence, cataracts, and other symptoms. The pathogenic effect of expanded CTG repeats more ..
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 Tested Compounds
 Tested Compounds
All(279434)
 
 
Active(99)
 
 
Inactive(248806)
 
 
Inconclusive(29679)
 
 
Unspecified(1175)
 
 
 Tested Substances
 Tested Substances
All(279988)
 
 
Active(100)
 
 
Inactive(248993)
 
 
Inconclusive(29714)
 
 
Unspecified(1181)
 
 
AID: 2675
Data Source: NCGC (MBNL923)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-25
Modify Date: 2014-02-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 99
Related Experiments
AIDNameTypeComment
2700qHTS Assay for Inhibitors of MBNL1-poly(CUG) RNA binding: SummarySummarydepositor-specified cross reference
493199qHTS Assay for Inhibitors of MBNL1-poly(CUG) RNA binding: Initial hit validation from the primary screenConfirmatorydepositor-specified cross reference
493205qHTS Assay for Inhibitors of MBNL1-poly(CUG) RNA binding: Initial hit validation in AlphaScreen assay for MBNL1-(CUG)12 bindingConfirmatorydepositor-specified cross reference
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLPCN Grant: R03 MH087421-01
Assay Submitter (PI): Charles Thornton

NCGC Assay Overview:

Expanded CTG repeats in the 3' untranslated region of the DM protein kinase (DMPK) gene causes type I myotonic dystrophy (DM1). DM1 is a multi-systemic disease with a prevalence of 1/7000. Clinical features include myotonia, progressive muscle weakness and wasting, cardiac arrhythmia, insulin resistance, hypersomnolence, cataracts, and other symptoms. The pathogenic effect of expanded CTG repeats stems from deleterious effects of the mutant transcript. CUGexp mRNA accumulates in nuclear foci and sequesters proteins of the muscleblind family, of which MBNL1 is the most common family member in skeletal muscles. The binding interaction between MBNL1 and CUGexp tracts leads to deregulation of alternative splicing and is therefore a key molecular step in the pathogenesis of DM1.

To target this binding interaction, we optimized a protein-RNA binding assay in 1536-well format for identification of small molecule inhibitors of biotinylated (CUG)12 and his-tagged MBNL1 binding. Protein-RNA complex of MBNL1-His and Biotin-(CUG)12 captures the detection reagents terbium-labeled anti-His antibody and streptavidin conjugated XL665. This brings the terbium label within close proximity to XL665 to allow time-resolved fluorescence resonance energy transfer (TR-FRET). Inhibitors of MBNL1-(CUG)12 binding will disrupt TR-FRET.
Protocol
NCGC Assay Protocol Summary:

This is a TR-FRET binding assay with MBNL1-His and Biotin-(CUG)12 RNA. The assay uses a ratiometric measurement. In the presence of MBNL1-(CUG)12 complex, the excitation energy is transferred to the XL665 acceptor label, causing emission at 665nm, while the 545nm donor signal remains the same. Data were normalized to the controls for basal activity (buffer only) and 100% activity (with MBNL1-(CUG)12). The IC50 values were determined from concentration-response data modeled with the standard Hill equation

Assay buffer: 20mM HEPES, 110mM KCl, 10mM NaCl, 2mM MgCl2, 2mM CaCl2, 5mM DTT, 0.1% BSA, 0.05% Tween 20


1536-well assay protocol for the MBNL1-(CUG)12 binding assay:
(1) Add 2 ul/well 20nM MBNL1-His, 20nM Biotin-(CUG)12
(2) Add 23 nL compounds in DMSO solution. The final titration was 92 nM to 58 uM.
(3) Add 1 ul of terbium labeled anti-His antibody (0.055 ng/ul final concentration)
(4) Add 1 ul of streptavidin-XL665 (5 nM final concentration)
(5) Incubate 60 min at room temperature
(6) Detect the assay plate in a EnVision plate reader (PerkinElmer) with Ex=340 nm, Em1=665 nm and Em2=545 nm.

Keywords: mytonic dystrophy type I, DM1, muscleblind like 1, MBNL1, CUG expansion, protein-RNA binding inhibitor, high throughput screening, MLSMR, MLSCN, 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 "Ratio-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
5Ratio-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 significant, but below 80% of control.String
6Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Ratio-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
13Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Ratio-Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
15Ratio-Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
16Ratio-Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
17Ratio-Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
18Ratio-Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
19Ratio-Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
20Donor-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 significant, but below 80% of control.String
21Donor-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
22Donor-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
23Donor-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
24Donor-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
25Donor-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
26Donor-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
27Donor-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
28Donor-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
29Donor-Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
30Donor-Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
31Donor-Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
32Donor-Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
33Donor-Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
34Donor-Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
35Donor-Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH087421-01

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