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BioAssay: AID 690

Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphatase

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified: three isozymes are tissue-specific and the fourth one is tissue-nonspecific. Placental alkaline phosphatase (PLAP) is highly expressed in primate placental tissue. Its biological function is unknown. Identification of PLAP-specific inhibitors should provide necessary tools for characterization of its biological role. ..more
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 Tested Compounds
 Tested Compounds
All(95856)
 
 
Active(82)
 
 
Inactive(95774)
 
 
 Tested Substances
 Tested Substances
All(95864)
 
 
Active(82)
 
 
Inactive(95782)
 
 
AID: 690
Data Source: Burnham Center for Chemical Genomics (SDCCG-A022-PLAP-inhibitors)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-04-23
Modify Date: 2010-08-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 82
Related Experiments
AIDNameTypeProbeComment
518TNAP luminescent HTS assayConfirmatory depositor-specified cross reference
1512Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphatase confirmationConfirmatory depositor-specified cross reference
1577Summary luminescent assay for discovery of chemical inhibitors of placental alkaline phosphataseSummary2 depositor-specified cross reference
696Luminescent assay for HTS discovery of chemical activators of placental alkaline phosphataseConfirmatory same project related to Summary assay
1017Luminescent assay for identification of inhibitors of human intestinal alkaline phosphataseConfirmatory same project related to Summary assay
1019Luminescent assay for identification of inhibitors of bovine intestinal alkaline phosphataseScreening same project related to Summary assay
1056SAR analysis of an In Vitro TNAP Dose Response Luminescent AssayConfirmatory same project related to Summary assay
Description:
Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified: three isozymes are tissue-specific and the fourth one is tissue-nonspecific. Placental alkaline phosphatase (PLAP) is highly expressed in primate placental tissue. Its biological function is unknown. Identification of PLAP-specific inhibitors should provide necessary tools for characterization of its biological role.

PLAP screening was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). This assay represents a selectivity screening for tissue nonspecific alkaline phosphatase (TNAP) screened at BCCG (AID 518). XO1 submission, MH077602-01, Pharmacological inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), Assay Provider Dr. Jose Luis Millan, Sanford-Burnham Medical Research Institute, San Diego, CA.
Protocol
PLAP HTS protocol:
PLAP assay materials:
1) PLAP protein was provided by Dr. Jose Luis Millan (Sanford-Burnham Medical Research Institute, San Diego, CA). The CDP-star was obtained from New England Biolabs.
2) Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl.
3) PLAP working solution contained a 1/6400 dilution in assay buffer. Solution was prepared fresh prior to use.
4) CDP-star working solution contained 212.5 uM CDP-star in MQ water.
5) TCEP working solution - 5 mM in 10% DMSO.
PLAP HTS protocol:
1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well white small volume plates (784075).
2) Using the Thermo wellmate dispenser 4 uL the following solutions were added:
a. TCEP working solution - column 1 (positive control).
b. 10% DMSO - column 2 (negative control).
3) 8 uL of PLAP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4) 8 uL of CDP-star working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
5) Final concentrations of the components in the assay were as follows:
a. 100 mM DEA, pH 9.8, 1.0 mM MgCl2, 0.02 mM ZnCl (columns 1-24)
b. 1/16000 dilution PLAP (columns 1-24)
c. 85 uM CDP-star (columns 1-24)
d. 1 mM TCEP (columns 1)
e. 2 % DMSO (columns 1-24)
f. 20 uM compounds (columns 3-24)
6) Plates were incubated for 30 mins at room temperature.
7) Luminescence was measured on the Envision plate reader (Perkin Elmer).
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
PLAP dose-response confirmation screening protocol:
1) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well white small-volume plates (784075). Columns 1-2 and 23-24 contained 4 uL of TCEP working solution and 10% DMSO, respectively.
2) 8 uL of PLAP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
3) 8 uL of CDP-star working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4) Plates were incubated for 30 mins at room temperature.
5) Luminescence was measured on the Envision plate reader (Perkin Elmer).
6) Data analysis was performed using CBIS software (ChemInnovations, Inc) using sigmoidal dose-response equation through non-linear regression
Comment
Compounds with greater than 50% inhibition of PLAP at 20-uM concentration are defined as actives of the primary screening. The primary screening actives proceed to the dose-response confirmation stage. Compounds that demonstrate IC50 values in the range of analyzed concentrations remain 'active' in the outcome column. Compounds that failed dose-response confirmation are assigned IC50 values equal to 999 (uM) and downgraded to 'inactive' in the outcome field.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the PLAP assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the PLAP assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data # the score is correlated with % displacement in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the PLAP assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*IC50 value determined using sigmoidal dose response equationFloatμM
2Std.Err(IC50)Standard Error of IC50 valueFloatμM
3nHHill coefficient determined using sigmoidal dose response equationFloat
4%Inhibition at 20 uM% inhibition of PLAP in primary screeningFloat
5Mean HighMean luminescence signal of negative controls in the corresponding plateFloatCPS
6STD Deviation HighStandard deviation (n=16) of negative controls in the corresponding plateFloatCPS
7Mean LowMean luminescence signal of positive controls in the corresponding plateFloatCPS
8STD Deviation LowStandard deviation (n=16) of positive controls in the corresponding plateFloatCPS

* Activity Concentration.

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