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BioAssay: AID 624044

S16 Schwann cell PMP22 intronic element beta-lactamase assay

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(2791)
 
 
Active(118)
 
 
Inactive(2244)
 
 
Inconclusive(432)
 
 
 Tested Substances
 Tested Substances
All(2814)
 
 
Active(119)
 
 
Inactive(2262)
 
 
Inconclusive(433)
 
 
AID: 624044
Data Source: NCGC (cmt-p4-fda-blac)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-10
Hold-until Date: 2012-04-30
Modify Date: 2012-04-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 118
Related Experiments
AIDNameTypeComment
624036Inhibitors for the S16 Schwann cell PMP22 intronic elementSummarydepositor-specified cross reference
624030Biochemical firefly luciferase enzyme assay for NPCConfirmatorysame project related to Summary assay
624031S16 Schwann cell viability assay (CellTiter-Glo assay)Confirmatorysame project related to Summary assay
624032S16 Schwann cell PMP22 intronic element firefly luciferase assayConfirmatorysame project related to Summary assay
Description:
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. The current assay employs Blac as a reporter of PMP22 expression. 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
S16 Schwann cell PMP22 intronic element beta-lactamase assay
The beta-Lac-expressing S16 cells were seeded in black clear-bottom 1536 well plates at between 500-750 cells/6uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then one microl of LiveBLAzer FRET B/G detection mix (Invitrogen) was added to each well. An EnVision plate reader (PerkinElmer) was used to detect the dual beta-Lac output composed of fluorescence at 460nm for beta-Lac expression and FRET at 535nm for cell viability. The % signal at 535 nm was scaled to a full range of DMSO and zero and then normalized using the maximum response observed in the screen as 100% activity. The % signal at 460nm was normalized to the average readings of potassium clavulanate (a positive control) - treated cells relative to DMSO-treated cells (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 was given for each active curve class type. Active compounds have PUBCHEM_ACTIVITY_SCORE between 70 and 100. Inconclusive compounds, and compounds found as active in the cytotoxicity channel have PUBCHEM_ACTIVITY_SCORE of 10 and 20, respectively."
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: S16
From ChEMBL:
Assay Type: Functional
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
5W460-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
6W460-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7W460-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8W460-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9W460-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10W460-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11W460-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12W460-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
13W460-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14W460-Activity at 0.0001100000 uM (0.00011μM**)% Activity at given concentration.Float%
15W460-Activity at 0.0004900000 uM (0.00049μM**)% Activity at given concentration.Float%
16W460-Activity at 0.0005480000 uM (0.000548μM**)% Activity at given concentration.Float%
17W460-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
18W460-Activity at 0.00254 uM (0.00254309μM**)% Activity at given concentration.Float%
19W460-Activity at 0.00548 uM (0.00548μM**)% Activity at given concentration.Float%
20W460-Activity at 0.013 uM (0.01275μM**)% Activity at given concentration.Float%
21W460-Activity at 0.027 uM (0.0274μM**)% Activity at given concentration.Float%
22W460-Activity at 0.064 uM (0.0636117μM**)% Activity at given concentration.Float%
23W460-Activity at 0.137 uM (0.137μM**)% Activity at given concentration.Float%
24W460-Activity at 0.318 uM (0.317867μM**)% Activity at given concentration.Float%
25W460-Activity at 0.711 uM (0.711001μM**)% Activity at given concentration.Float%
26W460-Activity at 1.588 uM (1.58779μM**)% Activity at given concentration.Float%
27W460-Activity at 3.507 uM (3.50651μM**)% Activity at given concentration.Float%
28W460-Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
29W460-Activity at 17.10 uM (17.1μM**)% Activity at given concentration.Float%
30W460-Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
31Ratio-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
32Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
33Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
34Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
35Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
36Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
37Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
38Ratio-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
39Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
40Ratio-Activity at 0.0001100000 uM (0.00011μM**)% Activity at given concentration.Float%
41Ratio-Activity at 0.0004900000 uM (0.00049μM**)% Activity at given concentration.Float%
42Ratio-Activity at 0.0005480000 uM (0.000548μM**)% Activity at given concentration.Float%
43Ratio-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
44Ratio-Activity at 0.00254 uM (0.00254309μM**)% Activity at given concentration.Float%
45Ratio-Activity at 0.00548 uM (0.00548μM**)% Activity at given concentration.Float%
46Ratio-Activity at 0.013 uM (0.01275μM**)% Activity at given concentration.Float%
47Ratio-Activity at 0.027 uM (0.0274μM**)% Activity at given concentration.Float%
48Ratio-Activity at 0.064 uM (0.0636117μM**)% Activity at given concentration.Float%
49Ratio-Activity at 0.137 uM (0.137μM**)% Activity at given concentration.Float%
50Ratio-Activity at 0.318 uM (0.317867μM**)% Activity at given concentration.Float%
51Ratio-Activity at 0.711 uM (0.711001μM**)% Activity at given concentration.Float%
52Ratio-Activity at 1.588 uM (1.58779μM**)% Activity at given concentration.Float%
53Ratio-Activity at 3.507 uM (3.50651μM**)% Activity at given concentration.Float%
54Ratio-Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
55Ratio-Activity at 17.10 uM (17.1μM**)% Activity at given concentration.Float%
56Ratio-Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
57W530-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
58W530-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
59W530-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
60W530-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
61W530-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
62W530-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
63W530-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
64W530-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
65W530-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
66W530-Activity at 0.0001100000 uM (0.00011μM**)% Activity at given concentration.Float%
67W530-Activity at 0.0004900000 uM (0.00049μM**)% Activity at given concentration.Float%
68W530-Activity at 0.0005480000 uM (0.000548μM**)% Activity at given concentration.Float%
69W530-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
70W530-Activity at 0.00254 uM (0.00254309μM**)% Activity at given concentration.Float%
71W530-Activity at 0.00548 uM (0.00548μM**)% Activity at given concentration.Float%
72W530-Activity at 0.013 uM (0.01275μM**)% Activity at given concentration.Float%
73W530-Activity at 0.027 uM (0.0274μM**)% Activity at given concentration.Float%
74W530-Activity at 0.064 uM (0.0636117μM**)% Activity at given concentration.Float%
75W530-Activity at 0.137 uM (0.137μM**)% Activity at given concentration.Float%
76W530-Activity at 0.318 uM (0.317867μM**)% Activity at given concentration.Float%
77W530-Activity at 0.711 uM (0.711001μM**)% Activity at given concentration.Float%
78W530-Activity at 1.588 uM (1.58779μM**)% Activity at given concentration.Float%
79W530-Activity at 3.507 uM (3.50651μM**)% Activity at given concentration.Float%
80W530-Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
81W530-Activity at 17.10 uM (17.1μM**)% Activity at given concentration.Float%
82W530-Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
83Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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