Bookmark and Share
BioAssay: AID 1136

uHTS identification of compounds activating TNAP at a high concentration of phosphate acceptor detected in a luminescent assay

Alkaline phosphatases (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organisms. In humans, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-non specific, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there is therapeutic potential of modulating TNAP activity. ..more
_
   
 Tested Compounds
 Tested Compounds
All(195554)
 
 
Active(5)
 
 
Inactive(195549)
 
 
 Tested Substances
 Tested Substances
All(195624)
 
 
Active(5)
 
 
Inactive(195619)
 
 
AID: 1136
Data Source: Burnham Center for Chemical Genomics (SDCCG-A054-TNAP Activator (Luminescent Assay at High DEA Con..)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-03-04
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
Show more
AIDNameTypeProbeComment
518TNAP luminescent HTS assayConfirmatory depositor-specified cross reference
813HTS identification of compounds activating TNAP at intermediate concentration of phosphate acceptor detected in luminescent assayScreening depositor-specified cross reference
1001uHTS identification of compounds activating TNAP in the absence of phosphate acceptor performed in luminescent assayConfirmatory depositor-specified cross reference
1548Summary assay for compounds activating TNAP performed in a luminescent assaySummary2 depositor-specified cross reference
1056SAR analysis of an In Vitro TNAP Dose Response Luminescent AssayConfirmatory same project related to Summary assay
1227GAPDH Dose Response Colorimetric AssayConfirmatory same project related to Summary assay
1450SAR assay for compounds activating TNAP in the absence of phosphate acceptor performed in a luminescent assayConfirmatory same project related to Summary assay
1659SAR assay for compounds activating TNAP in the presence of 100 mM DEA performed in a luminescence assayConfirmatory same project related to Summary assay
1941SAR assay for compounds inhibiting TNAP in the absence of phosphate acceptor performed in a luminescent assayConfirmatory same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Proposal Number: R03 MH082385-01

Assay Provider: Dr. Jose Luis Millan, Sanford-Burnham Medical Research Institute, San Diego, CA

Alkaline phosphatases (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organisms. In humans, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-non specific, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there is therapeutic potential of modulating TNAP activity.

TNAP luminescent assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG), part of the Molecular Library Screening Center Network (MLSCN). The goal of this HTS is to identify activators of TNAP with a novel mode of action. The only known to date class of alkaline phosphatases activators are nitrogen-containing alcohols, such as diethanolamine (DEA), that act as a phosphoacceptor substrate and exhibit its effect in high-mM concentration range. Identification of potent compounds mimicking the effect of the phosphoacceptors was addressed in a previous screening campaign (AID 1001). The current assay was specifically optimized and performed for identification of compounds with a different mechanism of action. To this end, we utilized a saturating concentration of DEA of 1 M in the assay: compounds with a mode of action similar to DEA are expected to demonstrate diminished stimulating potential if tested in the presence of DEA.
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 New England Biolabs.
2) Assay Buffer: 2 M DEA, pH 9.8, 2 mM MgCl2, and 0.04 mM ZnCl2.
3) TNAP working solution contained a 1/400 dilution in assay buffer.
4) CDP-star working solution contained 200 uM CDP-star in MQ water.
5) Negative Control (NC) solution: 500 mM EDTA
6) Positive Control (PC) solution: 100% DMSO
TNAP HTS protocol:
1) 2 uL of CDP-star working solution was added to all the well of Costar 1536-well white plate (cat #3725) using a Thermo Multidrop Combi dispenser
2) 40 nL of NC solution was added to columns 1-2 using a HighRes biosolutions pintool and V&P Scientific pins
3) 40 nL of PC solution was added to columns 3-4 using a HighRes biosolutions pintool and V&P Scientific pins
3) 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5-48 a HighRes biosolutions pintool and V&P Scientific pins
4) 2 uL of TNAP working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
5) Final concentrations of the components in the assay were as follows:
a. 1 M DEA, pH 9.8, 1.0 mM MgCl2, 0.02 mM ZnCl2
b. 1/800 dilution TNAP
c. 100 uM CDP-star
d. 20 uM compounds
6) Plates were incubated for 30 mins at room temperature.
7) Luminescence was measured on a ViewLux plate reader (Perkin Elmer).
8) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
9) Data analysis was performed using CBIS software (ChemInnovations, Inc).
Comment
TNAP activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC),
where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate.
Compounds with greater than or equal to 2-fold activation (AF >= 2) of TNAP at 20-uM concentration are defined as actives of the primary screening.
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 TNAP activation factor demonstrated by a compound at 20 uM concentration:
a. If AF<1, then the assigned score is 0
b. For all other AF values,
Score = 40 - 40/AF
This formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50EC50value determined using sigmoidal dose response equationFloatμM
2Std.Err(EC50)Standard Error of EC50 valueFloatμM
3nHHill coefficient determined using sigmoidal dose response equationFloat
4Max_ValueThe AF value asymptotically approached by the dose response curve at saturating compound concentrationsFloat
5AF_20uM (20μM**)% inhibition of TNAP in primary screeningFloat
6Mean_NCMean luminescence signal of negative controls in the corresponding plateFloatCPS
7StdDev_NCStandard deviation (n=64) of negative controls in the corresponding plateFloatCPS
8Mean_PCMean luminescence signal of positive controls in the corresponding plateFloatCPS
9StdDev_PCStandard deviation (n=64) of positive controls in the corresponding plateFloatCPS

** Test Concentration.

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