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BioAssay: AID 434971

Single concentration confirmation of uHTS hits from a small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assay

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(10803)
 
 
Active(509)
 
 
Inactive(10294)
 
 
 Tested Substances
 Tested Substances
All(10803)
 
 
Active(509)
 
 
Inactive(10294)
 
 
AID: 434971
Data Source: Burnham Center for Chemical Genomics (BCCG-A372-Mouse_IAP_Inhibitor-CP-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-06-03
Modify Date: 2011-01-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 509
Related Experiments
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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
1016Luminescent assay for identification of activators of bovine intestinal alkaline phosphataseScreeningdepositor-specified cross reference
2806uHTS Luminescent assay for identification of inhibitors of mouse intestinal alkaline phosphataseScreeningdepositor-specified cross reference
2818Summary assay for identification of inhibitors of mouse intestinal alkaline phosphataseSummarydepositor-specified cross reference
488785Dose Response confirmation of uHTS hits from a small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assayConfirmatorysame project related to Summary assay
488876Dose Response confirmation of uHTS inhibitors of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline PhosphataseConfirmatorysame project related to Summary assay
488879Dose Response confirmation of uHTS inhibitors of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline PhosphataseConfirmatorysame project related to Summary assay
488906Dose Response confirmation of uHTS inhibitors of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.Confirmatorysame project related to Summary assay
493135SAR analysis of small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assayConfirmatorysame project related to Summary assay
493136SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline PhosphataseConfirmatorysame project related to Summary assay
493144SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline PhosphataseConfirmatorysame project related to Summary assay
493148SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.Confirmatorysame project related to Summary assay
588639SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase - Set 2Confirmatorysame project related to Summary assay
588641SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline Phosphatase - Set 2Confirmatorysame project related to Summary assay
588645SAR analysis of small molecule inhibitors of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline Phosphatase - Set 2Confirmatorysame project related to Summary assay
588647SAR analysis of small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assay - Set 2Confirmatorysame 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 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.

The goal of this assay is to confirm hits in "uHTS Luminescent assay for identification of inhibitors of mouse intestinal alkaline phosphatase" (AID 2806) inorder to identify novel and specific inhibitors of mouse IAP. 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 MLPCN probe project is to obtain novel chemical scaffolds that can be used as chemical probes.
Protocol
Materials:
1. muIAP - 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 muIAP (1:125 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 (400 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 were tested in duplicate and those with % Inhibition of >= 40 % at 10 uM concentration for at least one replicate are defined as active 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 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 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:
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
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 10 uM_1 (10μM**)The % Inhibition at 10 uM for the first replicateFloat%
2%Inhibition at 10 uM_2 (10μM**)The % Inhibition at 10 uM for the second replicateFloat%
3Value_1 (10μM**)Value of the first replicateIntegerCPS
4Value_2 (10μM**)Value of the second replicateIntegerCPS
5Mean Low_1Mean luminescence signal of positive controls in the corresponding plate for the first replicateFloatCPS
6Mean Low_2Mean luminescence signal of negative controls in the corresponding plate for the second replicateFloatCPS
7Std Deviation Low_1Mean luminescence signal of positive controls in the corresponding plate for the first replicateFloatCPS
8Std Deviation Low_2Standard deviation (n=64) of negative controls in the corresponding plate for the second replicateFloatCPS
9Mean High_1Mean luminescence signal of positive controls in the corresponding plate for the first replicateFloatCPS
10Mean High_2Mean luminescence signal of positive controls in the corresponding plate for the second replicateFloatCPS
11Std Deviation High_1Standard deviation (n=64) of positive controls in the corresponding plate for the first replicateFloatCPS
12Std Deviation High_2Standard deviation (n=64) of positive controls in the corresponding plate for the second replicateFloatCPS

** Test Concentration.
Additional Information
Grant Number: X01-MH077602-01

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