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BioAssay: AID 602200

qHTS for Inhibitors of the vitamin D receptor (VDR): Hit Validation using a Fluorescein Assay

The vitamin D receptor (VDR) is a ligand-activated transcription factor and is partially responsible for the regulation of the endocrine system. This includes the transcriptional regulation of genes involved in the production of the parathyroid hormone (PTH) and the regulation of the blood calcium level. High levels of PHT can cause hypercalcemia in the case of primary and tertiary more ..
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 Tested Compounds
 Tested Compounds
All(821)
 
 
Active(390)
 
 
Inactive(47)
 
 
Inconclusive(387)
 
 
 Tested Substances
 Tested Substances
All(835)
 
 
Active(399)
 
 
Inactive(47)
 
 
Inconclusive(389)
 
 
AID: 602200
Data Source: NCGC (VDR202)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-18

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 390
Depositor Specified Assays
AIDNameTypeComment
504855Inhibitors of the vitamin D receptor (VDR): SummarysummarySummary
Description:
The vitamin D receptor (VDR) is a ligand-activated transcription factor and is partially responsible for the regulation of the endocrine system. This includes the transcriptional regulation of genes involved in the production of the parathyroid hormone (PTH) and the regulation of the blood calcium level. High levels of PHT can cause hypercalcemia in the case of primary and tertiary hyperparathyroidism. The VDR-mediated gene regulation, activated by its ligand 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is governed by the recruitment of coactivators. Our hypothesis is that small molecules, with the ability to inhibit the interaction between VDR and certain coactivators, are able to selectively modulate VDR-mediated transcription. Our long term objective is the application these VDR-coactivator inhibitors to study the role of VDR-coactivator binding for specific VDR regulated genes. Ultimately, these molecules can be developed into new treatments for hypercalcemic hyperparathyroidism. No small molecule or non-peptide inhibitors are known for the VDR-coactivator interaction.

In a collaboration between the University of Wisconsin, Milwaukee and the NIH Chemical Genomics Center (NCGC) a HTS-compatible enzymatic assay was developed. The assay uses fluorescence polarization as its read-out to measure the inhibition between VDR and coregulator peptide SRC2-3 exerted by small molecules. This assay was used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR). The HTS assay detects polarization at 665 nm emitted by the Alexa Fluor 647 dye. This validation assay measures polarization of fluorescein emission at 521 nm

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: DA031090
Assay Submitter (PI): Alexander Arnold, University of Wilwaukee
Protocol
Four microliter of either 1.5 uM VDR-LBD, 5 uM LG19078, and 5 nM SRC2-3 Fluorescein (all final concentrations) in columns 1, 2, and 5 to 48 or 5 nM SRC2-3 Fluorescein in columns 3 and 4 are dispensed into black medium-binding solid bottom Greiner plates. Both solutions are kept at 4 deg C and protected from light and dispensed using a Kalypsys dispenser. Then 23 nl of compounds and controls are added with Kalypsys pintool. The control is 26.5 mM CBT358 (final concentration of 151uM) and the compounds' final range in concentration is 457 nM - 114 uM. Plate is centrifuged at 1000 RPM for 15 seconds to remove any residual bubbles. The plate is then incubate at ambient room temperature for 2 hours. It is then read on the Envision for a fluorescent polarization read-out (Ex 495 nm/Em 520 nM)
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
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 significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the 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 0.091 uM (0.0914μM**)% Activity at given concentration.Float%
15Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
16Activity at 2.290 uM (2.29μM**)% Activity at given concentration.Float%
17Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
18Activity at 57.10 uM (57.1μM**)% Activity at given concentration.Float%
19Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA031090

Data Table (Concise)
Classification
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