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BioAssay: AID 1016

Luminescent assay for identification of activators of bovine intestinal alkaline phosphatase

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: more ..
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 Tested Compounds
 Tested Compounds
All(195574)
 
 
Active(326)
 
 
Inactive(195249)
 
 
 Tested Substances
 Tested Substances
All(195645)
 
 
Active(326)
 
 
Inactive(195319)
 
 
 Related BioAssays
 Related BioAssays
AID: 1016
Data Source: Burnham Center for Chemical Genomics (SDCCG-A039 Bovine IAP Activator)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-01-07
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 326
Related Experiments
AIDNameTypeComment
518TNAP luminescent HTS assayConfirmatorydepositor-specified cross reference
813HTS identification of compounds activating TNAP at intermediate concentration of phosphate acceptor detected in luminescent assayScreeningdepositor-specified cross reference
2524uHTS Luminescent assay for identification of activators of human intestinal alkaline phosphataseScreeningdepositor-specified cross reference
2805uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphataseScreeningdepositor-specified cross reference
2806uHTS Luminescent assay for identification of inhibitors of mouse intestinal alkaline phosphataseScreeningdepositor-specified cross reference
434926Single concentration confirmation of uHTS hits from a small molecule activators of human intestinal alkaline phosphatase via a luminescent assayScreeningdepositor-specified cross reference
434970Single concentration confirmation of uHTS hits from a small molecule activators of mouse intestinal alkaline phosphatase via a luminescent assayScreeningdepositor-specified cross reference
434971Single concentration confirmation of uHTS hits from a small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assayScreeningdepositor-specified cross reference
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: MH082385-01

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) alkaline phosphatases. IAP expression is largely restricted to the gut, especially to the epithelial cells (enterocytes) of the small intestinal mucosa. The exact biological function of IAP is unknown.

The goal of this HTS is to identify novel and specific activators of IAP. The only known to date class of alkaline phosphatases activators are hydroxyl-containing compounds, such as diethanolamine (DEA), that act as phosphoacceptor substrate and exhibit its effect in high-mM concentration range. Compounds with a similar mode of action are expected to demonstrate diminished stimulating potential if tested in the presence of high concentration of DEA. Therefore, for detection of compounds with diverse mode of action, the HTS campaign was performed in the presence close-to-Km DEA concentration.

IAP 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 activators of tissue nonspecific alkaline phosphatase (TNAP) screened at BCCG (AID 1001). RO3 submission, MH082385-01: Activators of the Pyrophosphatase Activity of Alkaline Phosphatase. Assay Providers: Drs. Jose Luis Millan and Eduard Sergienko, Sanford-Burnham Medical Research Institute, San Diego, CA.


Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) alkaline phosphatases. IAP expression is largely restricted to the gut, especially to the epithelial cells (enterocytes) of the small intestinal mucosa. The exact biological function of IAP is unknown.

IAP is inhibited by a number of inhibitors. They include L-phenylalanine, L-tryptophan, L-leucine and phenylalanine-glycylglycine. While the biological implications of this inhibition are not known, these inhibitors have proven to be useful in the differential determination of AP isozymes as important diagnostic markers in many diseases. However, these known inhibitors of IAP are not entirely specific for IAP isozyme and have milllimolar affinity. In addition, they are common aminoacids that are ubiquitously present in the tissues and involved in diverse metabolic pathways, and therefore, are not appropriate tools for biological studies. Thus, the aim of this MLSCN probe project is to obtain novel chemical scaffolds that can be used as chemical probes for IAP. Bovine enzyme was utilized in this primary screening as a highly active alternative with high homology to the human enzyme.

IAP screening was designed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). The assay was developed as a secondary assay for TNAP probe generation project (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
Materials:
1)Bovine IAP protein and CDP-star substrate were obtained from Biozyme Laboratories and Applied Biosystems, respectively.
2)Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl2.
3)IAP working solution contained a 1/26,000,000 dilution in assay buffer.
4)CDP-star working solution contained 75 uM CDP-star in MQ water.
IAP primary 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 the following solutions were added:
a.4 uL of 10% DMSO were added to columns 1-2
b.8 uL of water was added to column 1
c.8 uL of IAP working solution was added to column 1-24
d.8 uL of CPD-star was added to columns 2-24
3)Negative Control (NC) solution: no substrate samples (columns 1).
4)Positive Control (PC) solution: both the substrate and enzyme present (columns 3).
5)Plates were incubated for 30 mins at room temperature.
6)Luminescence was measured on the Envision plate reader (Perkin Elmer).
7)Data analysis was performed using CBIS software (ChemInnovations, Inc).
Comment
IAP 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 IAP 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 IAP 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 IAP assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and the score is correlated with IAP 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
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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**)IAP Activation Factor in the primary screeningFloat
6Mean_NCMean luminescence signal of negative controls in the corresponding plateFloatCPS
7StdDev_NCStandard deviation (n=16) of negative controls in the corresponding plateFloatCPS
8Mean_PCMean luminescence signal of positive controls in the corresponding plateFloatCPS
9StdDev_PCStandard deviation (n=16) of positive controls in the corresponding plateFloatCPS

** Test Concentration.

Data Table (Concise)
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