SAR analysis of small molecule activators of Mouse Intestinal Alkaline Phosphatase using Placental Alkaline Phosphatase
Alkaline phosphatase (EC 18.104.22.168) (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 ..
BioActive Compounds: 16
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 22.214.171.124) (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.
This confirmatory, concentration-response assay has been developed and performed to study the structure-activity relationship of analogs of the confirmed hits from "uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphatase" (AID 2805) by testing their ortholog specificity. Compounds are either acquired from commercial sources or synthesized internally.
1. PLAP - provided by Dr. Jose Luis Millan
2. CDP-Star (New England Biolabs # N7001S)
3. PLAP buffer - 200 mM DEA, 0.04 mM ZnCl2, 2 mM MgCl2
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 PLAP (1:1000 dilution in PLAP buffer) (columns 3 through 48)
a. For negative control add 2 uL of PLAP buffer instead of PLAP to columns 1 and 2
3. Add 2 uL/well of CDP-Star (250 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
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 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)