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BioAssay: AID 518

TNAP luminescent HTS assay

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organism. In human, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-nonsepecifc, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there are therapeutic potentials of inhibiting TNAP activity. The goal of this HTS is to identify novel and specific inhibitors of TNAP. ..more
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 Tested Compounds
 Tested Compounds
All(64387)
 
 
Active(54)
 
 
Inactive(64333)
 
 
 Tested Substances
 Tested Substances
All(64394)
 
 
Active(54)
 
 
Inactive(64340)
 
 
AID: 518
Data Source: Burnham Center for Chemical Genomics (SDCCG-A011-TNAP)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-11-06
Modify Date: 2010-08-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 54
Related Experiments
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AIDNameTypeProbeComment
614HTS colorimetric detection of phosphate released in TNAP reactionScreening depositor-specified cross reference
615HTS colorimetric detection of p-nitrophenol released in TNAP reactionScreening depositor-specified cross reference
690Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphataseConfirmatory depositor-specified cross reference
696Luminescent assay for HTS discovery of chemical activators of placental alkaline phosphataseConfirmatory depositor-specified cross reference
1001uHTS identification of compounds activating TNAP in the absence of phosphate acceptor performed in luminescent assayConfirmatory depositor-specified cross reference
1012uHTS identification of TNAP inhibitors in the absence of phosphate acceptor performed in luminescent assayScreening depositor-specified cross reference
1016Luminescent assay for identification of activators of bovine intestinal alkaline phosphataseScreening depositor-specified cross reference
1017Luminescent assay for identification of inhibitors of human intestinal alkaline phosphataseConfirmatory depositor-specified cross reference
1019Luminescent assay for identification of inhibitors of bovine intestinal alkaline phosphataseScreening depositor-specified cross reference
1056SAR analysis of an In Vitro TNAP Dose Response Luminescent AssayConfirmatory depositor-specified cross reference
1135uHTS identification of compounds inhibiting TNAP at a high concentration of phosphate acceptor detected in a luminescent assayScreening depositor-specified cross reference
1136uHTS identification of compounds activating TNAP at a high concentration of phosphate acceptor detected in a luminescent assayScreening depositor-specified cross reference
1450SAR assay for compounds activating TNAP in the absence of phosphate acceptor performed in a luminescent assayConfirmatory depositor-specified cross reference
1512Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphatase confirmationConfirmatory depositor-specified cross reference
1548Summary assay for compounds activating TNAP performed in a luminescent assaySummary2 depositor-specified cross reference
1577Summary luminescent assay for discovery of chemical inhibitors of placental alkaline phosphataseSummary2 depositor-specified cross reference
1659SAR assay for compounds activating TNAP in the presence of 100 mM DEA performed in a luminescence assayConfirmatory depositor-specified cross reference
1941SAR assay for compounds inhibiting TNAP in the absence of phosphate acceptor performed in a luminescent assayConfirmatory depositor-specified cross reference
2524uHTS Luminescent assay for identification of activators of human intestinal alkaline phosphataseScreening depositor-specified cross reference
2544uHTS Luminescent assay for identification of inhibitors of human intestinal alkaline phosphataseScreening depositor-specified cross reference
2805uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphataseScreening depositor-specified cross reference
2806uHTS Luminescent assay for identification of inhibitors of mouse intestinal alkaline phosphataseScreening depositor-specified cross reference
434926Single concentration confirmation of uHTS hits from a small molecule activators of human intestinal alkaline phosphatase via a luminescent assayScreening depositor-specified cross reference
434927Single concentration confirmation of uHTS hits from a small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assayScreening depositor-specified cross reference
434970Single concentration confirmation of uHTS hits from a small molecule activators of mouse intestinal alkaline phosphatase via a luminescent assayScreening depositor-specified cross reference
434971Single concentration confirmation of uHTS hits from a small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assayScreening depositor-specified cross reference
813HTS identification of compounds activating TNAP at intermediate concentration of phosphate acceptor detected in luminescent assayScreening same project related to Summary assay
1227GAPDH Dose Response Colorimetric 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)
Grant Number: MH077602-01

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organism. In human, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-nonsepecifc, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there are therapeutic potentials of inhibiting TNAP activity. The goal of this HTS is to identify novel and specific inhibitors of TNAP.

TNAP screening was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). 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
TNAP assay protocol.
TNAP assay materials:
1) TNAP protein was provided by Dr. Jose Luis Millan (Sanford-Burnham Medical Research Institute, San Diego, CA). The CDP-star was obtained from Applied Biosystems.
2) Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl2.
3) TNAP working solution contained a 1/800 dilution in assay buffer. Solution was prepared fresh prior to use.
4) CDP-star working solution contained 125 uM CDP-star in MQ water.
5) Levamisole working solution - 5 mM in 10% DMSO.
TNAP 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. 10% DMSO - column 1 (negative control).
b. Levamisole working solution - columns 2 (positive control).
3) 8 uL of TNAP 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 ZnCl2 (columns 1-24)
b. 1/2000 dilution TNAP (columns 1-24)
c. 50 uM CDP-star (columns 1-24)
d. 1 mM Levamisole (columns 2)
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).
TNAP 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 levamisole working solution and 10% DMSO, respectively.
2) 8 uL of TNAP 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 TNAP 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.
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 TNAP 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 TNAP assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and 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's 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 TNAP 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
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50 *IC50 value determined using sigmoidal dose response equationFloatμM
3St.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%Inhibition at 20 uM% inhibition of TNAP in primary screeningFloat
6Mean HighMean luminescence signal of negative controls in the corresponding plateFloatCPS
7STD Deviation HighStandard deviation (n=16) of negative controls in the corresponding plateFloatCPS
8Mean LowMean luminescence signal of positive controls in the corresponding plateFloatCPS
9STD Deviation LowStandard deviation (n=16) of positive controls in the corresponding plateFloatCPS

* Activity Concentration.
Additional Information
Grant Number: MH077602-01

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