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BioAssay: AID 2544

uHTS Luminescent assay for identification of inhibitors of human 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(330397)
 
 
Active(393)
 
 
Inactive(330004)
 
 
 Tested Substances
 Tested Substances
All(330480)
 
 
Active(393)
 
 
Inactive(330087)
 
 
AID: 2544
Data Source: Burnham Center for Chemical Genomics (BCCG-A322-Human_IAP_Inhibitor-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-13
Modify Date: 2011-01-13

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 393
Depositor Specified Assays
Show more
AIDNameTypeComment
518TNAP luminescent HTS assayconfirmatory
463135Dose Response confirmation of uHTS hits from a small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assayconfirmatory
540274SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Mouse Intestinal Alkaline Phosphatase - Set 2confirmatory
488793Dose Response confirmation of uHTS hits from a small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assay - Set 2confirmatory
540265SAR analysis of small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assay - Set 2confirmatory
434927Single concentration confirmation of uHTS hits from a small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assayscreening
540260SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Placental Alkaline Phosphatase - Set 2confirmatory
488874Dose Response confirmation of uHTS inhibitors of Human Intestinal Alkaline Phosphatase using Mouse Intestinal Alkaline Phosphataseconfirmatory
488882Dose Response confirmation of uHTS inhibitors of Human Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.confirmatory
2574Summary assay for identification of inhibitors of human intestinal alkaline phosphatasesummary
493141SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.confirmatory
540261SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase - Set 2confirmatory
493137SAR analysis of small molecule inhibitors of human intestinal alkaline phosphatase via a luminescent assayconfirmatory
493138SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Placental Alkaline Phosphataseconfirmatory
488892Dose Response confirmation of uHTS inhibitors of Human Intestinal Alkaline Phosphatase using Placental Alkaline Phosphataseconfirmatory
493142SAR analysis of small molecule inhibitors of Human Intestinal Alkaline Phosphatase using Mouse Intestinal Alkaline Phosphataseconfirmatory
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 Probe Production Centers Network (MLPCN)
Grant Proposal Number: X01-MH077602-01
Assay Provider: Dr. Jose Luis Millan, 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 (1). They include L-phenylalanine, (2, 3), L-tryptophan (4), L-leucine and phenylalanine-glycylglycine (5). 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.
Protocol
Materials:
1. IAP - provided by Dr. Jose Luis Millan
2. CDP-Star (New England Biolabs # N7001S)
3. IAP buffer - 200 mM DEA, 0.04 mM ZnCl2, 2 mM MgCl2
Protocol
1. Add 30nl of 2mM compound in 100% DMSO to columns 5 to 48 in a 1536 well plate (Nexus/Aurora # 00029847)
2. Add 30nl of 100% DMSO to columns 1 through 4 (control wells)
3. Add 3 uL/well of IAP (1:200 dilution in IAP buffer) (columns 3 through 48)
a. For negative control add 3 uL of IAP buffer instead of IAP to columns 1 and 2
4. Add 3 uL/well of CDP-Star (354 uM in MQ water) to all wells
5. Spin the plate down to maintain an even level of volume
6. Cover the plate and incubate the plate at RT for 30 minutes
7. Read the plate on Perkin Elmer EnVision using US-Luminescence mode
Comment
Compounds that demonstrated % activity of >= 35 % at 10 uM concentration are defined as actives of the primary screen in this assay.

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 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 assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 10 uM (10μM**)% inhibition of IAP in primary screeningFloat%
2Mean HighMean luminescence signal of negative controls (n=64) in the corresponding plateFloatCPS
3STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatCPS
4Mean Low Mean luminescence signal of positive controls (n=64) in the corresponding platFloatCPS
5STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatCPS

** Test Concentration.
Additional Information
Grant Number: XO1 MH077602-01

Data Table (Concise)
Classification
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