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BioAssay: AID 624030

Biochemical firefly luciferase enzyme assay for NPC

Charcot-Marie-Tooth (CMT) disease was first characterized by Jean-Martin Charcot and Pierre Marie in France and, independently, by Howard Henry Tooth in England in 1886. CMT is one of the most common inherited neurological diseases, affecting approximately 1 in 2,500 Americans. CMT patients typically exhibit muscle atrophy in the extremities and sensory loss. The most prevalent type of CMT is more ..
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 Tested Compounds
 Tested Compounds
All(3609)
 
 
Active(104)
 
 
Inactive(3270)
 
 
Inconclusive(246)
 
 
 Tested Substances
 Tested Substances
All(3719)
 
 
Active(106)
 
 
Inactive(3358)
 
 
Inconclusive(255)
 
 
AID: 624030
Data Source: NCGC (adst-fluc-km-fda-o1_regid)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-07
Hold-until Date: 2012-04-30
Modify Date: 2012-04-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 104
Depositor Specified Assays
AIDNameTypeComment
624036Inhibitors for the S16 Schwann cell PMP22 intronic elementsummary
2309Probe Summary for Inhibitors and Stabilizers of Firefly Luciferasesummary
Description:
Charcot-Marie-Tooth-Association [CMTA]
National Center for Advancing Translational Sciences [NCATS]

Charcot-Marie-Tooth (CMT) disease was first characterized by Jean-Martin Charcot and Pierre Marie in France and, independently, by Howard Henry Tooth in England in 1886. CMT is one of the most common inherited neurological diseases, affecting approximately 1 in 2,500 Americans. CMT patients typically exhibit muscle atrophy in the extremities and sensory loss. The most prevalent type of CMT is known as CMT1A and caused by a duplication of the PMP22 gene which leads to over-expression of this protein. PMP22 is a glycosylated intrinsic membrane protein accounting for 2-5% of the myelin protein content, and its over-expression results in demyelination and subsequently axonal loss.

A pair of cellular transcription-based assays was developed in the S16 rat Schwann cell lines (Hai, M., et al., 2002) stably transfected with orthogonal reporters - firefly luciferase (Fluc) and beta-lactamase (beta-Lac) - whose expression is driven by an intronic regulatory element of the PMP22 gene (Jones, E.A., et al., 2011) to recapitulate endogenous expression of PMP22. A comprehensive library of approved drugs, the NCGC Pharmaceutical Collection (Huang, R., et al., 2011), was screened in the cross-validating platform of the orthogonal reporter assays to identify drug modulators inhibitory towards PMP22 expression.

A set of counter-screens was devised to address non-specific activity of the library. An ATP-dependent CellTiter-Glo assay (developed by Promega Corp) was utilized to control cell viability. In addition, biochemical Fluc enzyme assay was performed to assess direct inhibition of the Fluc enzymatic activity.
References
Hai, M., Muja, N., DeVries, G.H., Quarles, R.H. and Patel, P.I. Comparative analysis of Schwann cell lines as model systems for myelin gene transcription studies. J Neurosci Res, 2002. 69(4): pp 497-508
Huang, R., Southall, N., Wang, Y., Yasgar A., Shinn P., Jadhav, A. , Nguyen, D.-T. and Austin, C.P., The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Transl Med, 2011. 3(80): p. 80ps16.
Protocol
Three microliters/well of substrate- buffer solution (0.01 mM D-Luciferin, 0.01 mM ATP, 50 mM Tris Acetate, 10 mM Mg Acetate, 0.01% Tween-20 and 0.038% BSA final concentrations) were dispensed into white solid-bottom 1536 well plates. Compounds were transferred to the plates in 23nl, and one microliter/well luciferase enzyme-buffer solution (10 nM P. pyralis luciferase and 50 mM Tris Acetate final concentrations) was dispensed yielding a final reaction volume of 4 ul/well. Luciferase luminescence was measured by a ViewLux plate reader within 5 minutes of enzyme-buffer addition. The % activity was determined by normalizing to the average readings of PTC124 (a positive control) relative to DMSO (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.
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
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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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0001640000 uM (0.000164μM**)% Activity at given concentration.Float%
16Activity at 0.0007600859 uM (0.000760086μM**)% Activity at given concentration.Float%
17Activity at 0.00164 uM (0.00164μM**)% Activity at given concentration.Float%
18Activity at 0.00357 uM (0.00356794μM**)% Activity at given concentration.Float%
19Activity at 0.00764 uM (0.00764249μM**)% Activity at given concentration.Float%
20Activity at 0.016 uM (0.0161602μM**)% Activity at given concentration.Float%
21Activity at 0.019 uM (0.0186791μM**)% Activity at given concentration.Float%
22Activity at 0.041 uM (0.0407434μM**)% Activity at given concentration.Float%
23Activity at 0.084 uM (0.0836504μM**)% Activity at given concentration.Float%
24Activity at 0.098 uM (0.0979312μM**)% Activity at given concentration.Float%
25Activity at 0.204 uM (0.204029μM**)% Activity at given concentration.Float%
26Activity at 0.443 uM (0.442547μM**)% Activity at given concentration.Float%
27Activity at 0.601 uM (0.601348μM**)% Activity at given concentration.Float%
28Activity at 1.020 uM (1.02034μM**)% Activity at given concentration.Float%
29Activity at 2.221 uM (2.22135μM**)% Activity at given concentration.Float%
30Activity at 3.851 uM (3.85066μM**)% Activity at given concentration.Float%
31Activity at 5.200 uM (5.19999μM**)% Activity at given concentration.Float%
32Activity at 11.13 uM (11.1327μM**)% Activity at given concentration.Float%
33Activity at 24.87 uM (24.8652μM**)% Activity at given concentration.Float%
34Activity at 50.60 uM (50.6007μM**)% Activity at given concentration.Float%
35Activity at 58.59 uM (58.5873μM**)% Activity at given concentration.Float%
36Activity at 123.2 uM (123.162μM**)% Activity at given concentration.Float%
37Activity at 215.3 uM (215.252μM**)% Activity at given concentration.Float%
38Activity at 288.0 uM (288μM**)% Activity at given concentration.Float%
39Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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