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BioAssay: AID 493146

SAR analysis of small molecule activators 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(117)
 
 
Active(3)
 
 
Inactive(114)
 
 
 Tested Substances
 Tested Substances
All(120)
 
 
Active(3)
 
 
Inactive(117)
 
 
AID: 493146
Data Source: Burnham Center for Chemical Genomics (SBCCG-A564-Mouse_IAP_Activator-DryPowder-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-01
Hold-until Date: 2012-01-31
Modify Date: 2012-01-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeComment
2805uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphataseScreeningdepositor-specified cross reference: Primary Screen
2817Summary assay for identification of activators of mouse intestinal alkaline phosphataseSummarydepositor-specified cross reference: Summary AID.
434970Single concentration confirmation of uHTS hits from a small molecule activators of mouse intestinal alkaline phosphatase via a luminescent assayScreeningsame project related to Summary assay
488783Dose Response concentration confirmation of uHTS hits from a small molecule activators of mouse intestinal alkaline phosphatase via a luminescent assayConfirmatorysame project related to Summary assay
488873Dose Response confirmation of uHTS activators of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline PhosphataseConfirmatorysame project related to Summary assay
488878Dose Response confirmation of uHTS activators of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline PhosphataseConfirmatorysame project related to Summary assay
488880Dose Response confirmation of uHTS activators of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.Confirmatorysame project related to Summary assay
493133SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase.Confirmatorysame project related to Summary assay
493139SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline PhosphataseConfirmatorysame project related to Summary assay
493145SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline PhosphataseConfirmatorysame project related to Summary assay
588605SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Tissue Nonspecific Alkaline Phosphatase Set 2Confirmatorysame project related to Summary assay
588607SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline Phosphatase Set 2Confirmatorysame project related to Summary assay
588609SAR analysis of small molecule activators of mouse intestinal alkaline phosphatase via a luminescent assay Set 2Confirmatorysame project related to Summary assay
588611SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Human Intestinal Alkaline Phosphatase 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 Production Centers Network (MLPCN)
Grant Proposal Number: X01-MH077602-01

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 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.

The goal of this HTS is to confirm hits in "uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphatase" (AID 2805) inorder to identify novel and specific activators of mouse IAP and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
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. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 and 44-48 (Control wells), while varying volumes of test compounds are transferred to columns 4-44 to achieve the desired test concentrations. Compounds are transferred from a 10 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. Add 2 uL/well of muIAP (1:125 dilution in IAP buffer) (columns 3 through 48)
a. For negative control add 2 uL of IAP buffer instead of muIAP to columns 1 and 2
3. Add 2 uL/well of CDP-Star (400 uM in MQ water) to all wells
4. Spin the plate down to maintain an even level of volume
5. Cover the plate and incubate the plate at RT for 30 minutes
6. Read the plate on Perkin Elmer EnVision using US-Luminescence mode
Comment
Compounds with EC50_Mean < 100 uM defined as actives 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 and is not applicable in this assay.
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.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound 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 this 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 = 82 + 3*(pEC50 - 3)*QC
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Mean_QualifierThis qualifier is to be used with the next TID, EC50_Mean. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
2EC50_Mean*EC50 value determined using sigmoidal dose response equationFloatμM
3EC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4EC50_1EC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(EC50)_1Standard Error of EC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 100 uM_first_point (100μM**)% Activity at a given concentrationFloat%
9% Activity at 50 uM_first_point (50μM**)% Activity at a given concentrationFloat%
10% Activity at 25 uM_first_point (25μM**)% Activity at a given concentrationFloat%
11% Activity at 12.5 uM_first_point (12.5μM**)% Activity at a given concentrationFloat%
12% Activity at 6.25 uM_first_point (6.25μM**)% Activity at a given concentrationFloat%
13% Activity at 3.125 uM_first_point (3.125μM**)% Activity at a given concentrationFloat%
14% Activity at 1.5625 uM_first_point (1.5625μM**)% Activity at a given concentrationFloat%
15% Activity at 0.78125 uM_first_point (0.78125μM**)% Activity at a given concentrationFloat%
16% Activity at 0.390625 uM_first_point (0.390625μM**)% Activity at a given concentrationFloat%
17% Activity at 0.1953125 uM_first_point (0.195312μM**)% Activity at a given concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 100 uM_second_point (100μM**)% Activity at a given concentrationFloat%
20% Activity at 50 uM_second_point (50μM**)% Activity at a given concentrationFloat%
21% Activity at 25 uM_second_point (25μM**)% Activity at a given concentrationFloat%
22% Activity at 12.5 uM_second_point (12.5μM**)% Activity at a given concentrationFloat%
23% Activity at 6.25 uM_second_point (6.25μM**)% Activity at a given concentrationFloat%
24% Activity at 3.125 uM_second_point (3.125μM**)% Activity at a given concentrationFloat%
25% Activity at 1.5625 uM_second_point (1.5625μM**)% Activity at a given concentrationFloat%
26% Activity at 0.78125 uM_second_point (0.78125μM**)% Activity at a given concentrationFloat%
27% Activity at 0.390625 uM_second_point (0.390625μM**)% Activity at a given concentrationFloat%
28% Activity at 0.1953125 uM_second_point (0.195312μM**)% Activity at a given concentrationFloat%
29Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
30% Activity at 100 uM_third_point (100μM**)% Activity at a given concentrationFloat%
31% Activity at 50 uM_third_point (50μM**)% Activity at a given concentrationFloat%
32% Activity at 25 uM_third_point (25μM**)% Activity at a given concentrationFloat%
33% Activity at 12.5 uM_third_point (12.5μM**)% Activity at a given concentrationFloat%
34% Activity at 6.25 uM_third_point (6.25μM**)% Activity at a given concentrationFloat%
35% Activity at 3.125 uM_third_point (3.125μM**)% Activity at a given concentrationFloat%
36% Activity at 1.5625 uM_third_point (1.5625μM**)% Activity at a given concentrationFloat%
37% Activity at 0.78125 uM_third_point (0.78125μM**)% Activity at a given concentrationFloat%
38% Activity at 0.390625 uM_third_point (0.390625μM**)% Activity at a given concentrationFloat%
39% Activity at 0.1953125 uM_third_point (0.195312μM**)% Activity at a given concentrationFloat%
40EC50_Qualifier_2This qualifier is to be used with the next TID, EC50_2. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
41EC50_2EC50 value determined using sigmoidal dose response equationFloatμM
42Std.Err(EC50)_2Standard Error of EC50 valueFloatμM
43nH_2Hill coefficient determined using sigmoidal dose response equationFloat
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 100 uM_fourth_point (100μM**)% Activity at a given concentrationFloat%
46% Activity at 50 uM_fourth_point (50μM**)% Activity at a given concentrationFloat%
47% Activity at 25 uM_fourth_point (25μM**)% Activity at a given concentrationFloat%
48% Activity at 12.5 uM_fourth_point (12.5μM**)% Activity at a given concentrationFloat%
49% Activity at 6.25 uM_fourth_point (6.25μM**)% Activity at a given concentrationFloat%
50% Activity at 3.125 uM_fourth_point (3.125μM**)% Activity at a given concentrationFloat%
51% Activity at 1.5625 uM_fourth_point (1.5625μM**)% Activity at a given concentrationFloat%
52% Activity at 0.78125 uM_fourth_point (0.78125μM**)% Activity at a given concentrationFloat%
53% Activity at 0.390625 uM_fourth_point (0.390625μM**)% Activity at a given concentrationFloat%
54% Activity at 0.1953125 uM_fourth_point (0.195312μM**)% Activity at a given concentrationFloat%
55Excluded_Points_fifth_pointFlags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
56% Activity at 100 uM_fifth_point (100μM**)% Activity at a given concentrationFloat%
57% Activity at 50 uM_fifth_point (50μM**)% Activity at a given concentrationFloat%
58% Activity at 25 uM_fifth_point (25μM**)% Activity at a given concentrationFloat%
59% Activity at 12.5 uM_fifth_point (12.5μM**)% Activity at a given concentrationFloat%
60% Activity at 6.25 uM_fifth_point (6.25μM**)% Activity at a given concentrationFloat%
61% Activity at 3.125 uM_fifth_point (3.125μM**)% Activity at a given concentrationFloat%
62% Activity at 1.5625 uM_fifth_point (1.5625μM**)% Activity at a given concentrationFloat%
63% Activity at 0.78125 uM_fifth_point (0.78125μM**)% Activity at a given concentrationFloat%
64% Activity at 0.390625 uM_fifth_point (0.390625μM**)% Activity at a given concentrationFloat%
65% Activity at 0.1953125 uM_fifth_point (0.195312μM**)% Activity at a given concentrationFloat%
66Excluded_Points_sixth_pointFlags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
67% Activity at 100 uM_sixth_point (100μM**)% Activity at a given concentrationFloat%
68% Activity at 50 uM_sixth_point (50μM**)% Activity at a given concentrationFloat%
69% Activity at 25 uM_sixth_point (25μM**)% Activity at a given concentrationFloat%
70% Activity at 12.5 uM_sixth_point (12.5μM**)% Activity at a given concentrationFloat%
71% Activity at 6.25 uM_sixth_point (6.25μM**)% Activity at a given concentrationFloat%
72% Activity at 3.125 uM_sixth_point (3.125μM**)% Activity at a given concentrationFloat%
73% Activity at 1.5625 uM_sixth_point (1.5625μM**)% Activity at a given concentrationFloat%
74% Activity at 0.78125 uM_sixth_point (0.78125μM**)% Activity at a given concentrationFloat%
75% Activity at 0.390625 uM_sixth_point (0.390625μM**)% Activity at a given concentrationFloat%
76% Activity at 0.1953125 uM_sixth_point (0.195312μM**)% Activity at a given concentrationFloat%

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

Data Table (Concise)
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