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BioAssay: AID 607

qHTS Assay for Inhibitors of PDE-IV

The cyclic nucleotide phosphodiesterases (PDEs) are proteins that catalyze hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and have a central role in a variety of intracellular signaling events. cAMP PDEs are emerging as a promising class of drug targets in asthma and cardiovascular disease therapeutic areas. ..more
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 Tested Compounds
 Tested Compounds
All(8357)
 
 
Active(34)
 
 
Inactive(6951)
 
 
Inconclusive(1612)
 
 
 Tested Substances
 Tested Substances
All(9202)
 
 
Active(38)
 
 
Inactive(7498)
 
 
Inconclusive(1666)
 
 
 Related BioAssays
 Related BioAssays
AID: 607
Data Source: NCGC (PDE4426)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-03-02
Modify Date: 2007-11-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 34
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

NCGC Assay Overview:
The cyclic nucleotide phosphodiesterases (PDEs) are proteins that catalyze hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and have a central role in a variety of intracellular signaling events. cAMP PDEs are emerging as a promising class of drug targets in asthma and cardiovascular disease therapeutic areas.

There are no commercially available cell-based screening assays for PDE inhibitors: currently biochemical assays are done using pure substrates (cAMP or cGMP) and purified recombinant PDE enzymes. As with other biochemical screens on intracellular targets, false positives and false negatives may result from issues involving cell membrane permeability, cellular metabolism, and the like.

Here we report the development of a cell-based screening assay for cAMP phosphodiesterase (PDE) inhibitors. The assay is based on the BD ACTOne# technology (BD Biosciences) which utilizes a modified cyclic nucleotide-gated channel (CNG) to monitor intracellular cAMP changes in live cells. The CNG channel is localized on the plasma membrane, and opens when the intracellular cAMP level increases, resulting in ion flux and cell membrane depolarization. The influx of cations through the CNG channel can be quantified using membrane potential (MP) dyes. We modified the BD ACTOne technology to allow the detection of PDE inhibitors, and performed an inhibitor screen for PDE-IV, which is expressed endogenously in HEK293 cells. We optimized this assay in homogeneous 1536-well plate assay format for high throughput screening.

In order to help eliminate false positives, a counterscreen of the entire library using the parental cell line was conducted and compounds that showed signaling in both the parental and PDE assay cell line were labeled as 'Inconclusive'. Compounds which were active only in the PDE assay cell line were labeled as 'active' and compounds which signaled only in the parental cell line were labeled 'inactive'.
Protocol
NCGC Assay Protocol Summary:
ACTOneTM cells (HEK293 cells stably expressing a modified cyclic nucleotide-gated channel and optimized for screening PDE inhibitors) were maintained in DMEM (Invitrogen, Carlsbad, CA) at 37 degrees Celsius under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS (Invitrogen), 2 mM L-glutamine, and 250 ug/ml G418 (Cellgro) and 1 ug/ml puromycin (Clontech). ACTOneTM parental cells (BD Biosciences) were maintained in the same medium without puromycin.

The activity of the CNG channel was measured by BD ACTOneTM membrane potential dye kit (BD Biosciences). Forskolin, a positive control for cAMP activation and RO 20-1724, a PDE inhibitor, were both purchased from Sigma and solublized in DMSO.

Sequence,Parameter,Value,Description
1,Reagent,3 uL,1000 cells/well 1536 TC treated Black plate
2,Time,12 hours,37 C 5% CO2
3,Compound,0.5 uL,Kalypsys dispense RO 20-1724 100 uM final [A]
4,Reagent,3 uL,BD membrane potential dye
5,Time,1 hour,Room temp
6,Compound,23 nL,Final concentration was 0.7nM to 22.9uM
7,Time,1 hour,Room temp
8,Detector,EX 535 / EM 590 [B],Envision


Footnotes: [A] The stock 2 mM RO 20-1724 PDE inhibitor was made in 10 % DMSO. Parental cell line received 10 uM forskolin + 100 uM RO. [B] Envsion settings: Bottom Excitation 535/20 nm top emission 590/20, gain of 150 and 5 flashes per well.

Keywords: PDE, PDE-IV, cytotoxicity, flourescence, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
Comment
Compound Ranking:

1. Compounds are first classified as being probable activators (compounds causing an increase in membrane potential dye flourescence, presumably through inhibition of PDE), inhibitors (compounds causing a decrease in membrane porential dye flourescence), inactive, or inconclusive based on flourescence readout in ACTOne(TM) (pde) and parental cells.

2. Within the activators, compounds were ranked by efficacy and potency. The most potent and highest efficacy compounds were ranked highest, with a score of 99 to 40. Inhibitors were ranked with a score of 20, inconclusive with a rank of 10, and inactive with a rank of 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: signal activator, signal inhibitor, inactive, or inconclusive. Signal activators and inhibitors selectively affect flourescence of ACTOne(TM) cells.String
2PotencyConcentration 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.String
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
6pde-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7pde-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8pde-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9pde-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float
10pde-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float
11pde-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12pde-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
13parental-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
14parental-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
15parental-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
16parental-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float
17parental-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float
18parental-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
19parental-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
20pde-Activity at 0.30 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
21pde-Activity at 0.70 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
22pde-Activity at 1.5 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
23pde-Activity at 3.3 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
24pde-Activity at 7.4 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
25pde-Activity at 16.5 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
26pde-Activity at 36.8 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
27pde-Activity at 82.3 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
28pde-Activity at 183 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
29pde-Activity at 411 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
30pde-Activity at 919 nM% Activity of ACTOne (PDE) cells at given concentration.Float%
31pde-Activity at 2.05 uM% Activity of ACTOne (PDE) cells at given concentration.Float%
32pde-Activity at 4.59 uM% Activity of ACTOne (PDE) cells at given concentration.Float%
33pde-Activity at 10.2 uM% Activity of ACTOne (PDE) cells at given concentration.Float%
34pde-Activity at 22.9 uM% Activity of ACTOne (PDE) cells at given concentration.Float%
35parental-Activity at 0.4 nM% Activity of parental cells at given concentration.Float%
36parental-Activity at 0.8 nM% Activity of parental cells at given concentration.Float%
37parental-Activity at 1.8 nM% Activity of parental cells at given concentration.Float%
38parental-Activity at 4.1 nM% Activity of parental cells at given concentration.Float%
39parental-Activity at 9.2 nM% Activity of parental cells at given concentration.Float%
40parental-Activity at 20.6 nM% Activity of parental cells at given concentration.Float%
41parental-Activity at 46.0 nM% Activity of parental cells at given concentration.Float%
42parental-Activity at 102 nM% Activity of parental cells at given concentration.Float%
43parental-Activity at 229 nM% Activity of parental cells at given concentration.Float%
44parental-Activity at 514 nM% Activity of parental cells at given concentration.Float%
45parental-Activity at 1.14 uM% Activity of parental cells at given concentration.Float%
46parental-Activity at 2.57 uM% Activity of parental cells at given concentration.Float%
47parental-Activity at 5.74 uM% Activity of parental cells at given concentration.Float%
48parental-Activity at 12.8 uM% Activity of parental cells at given concentration.Float%
49parental-Activity at 28.7 uM% Activity of parental cells at given concentration.Float%
50Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
51Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
Classification
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