Luminescent assay for identification of inhibitors of human intestinal alkaline phosphatase
Alkaline phosphatase (EC 126.96.36.199) (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: 22
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: XO1 MH077602-01
Alkaline phosphatase (EC 188.8.131.52) (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.
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.
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.
1)Human IAP protein was provided by Dr. Jose Luis Millan (Sanford-Burnham Medical Research Institute, San Diego, CA). The CDP-star was obtained from Applied Biosystems.
2)Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl2.
3)IAP working solution contained a 1/80 dilution in assay buffer. Solution was prepared 1h prior to use and incubated at room temperature.
4)CDP-star working solution contained 442.5 uM CDP-star in MQ water.
5)L-phenylalanine working solution - 100 mM in 10% DMSO.
IAP dose-response confirmation screening protocol:
1)Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well white small-volume plates (784075). Each curve was performed in duplicate.
2)Columns 1-2 and 23-24 contained 4 uL of L-phenylalanine working solution and 10% DMSO, respectively.
3)8 uL of IAP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4)8 uL of CDP-star working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
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) using sigmoidal dose-response equation through non-linear regression
Representatives of the leading TNAP scaffold classes (see AID 518) were tested in the IAP assay.
The compounds were tested in 1 or more concentration ranges.
Range1 0-100 uM
Range2 0-10 uM
For all samples in each range that resolved to an IC50 the results were averaged and reported as IC50_Range. The IC50 results for all of the IC50s were averaged to produce IC50_Mean.
IC50 results are an average of two dose response curves. If for a given range there are 6 sets of curves there will be three IC50 results reported.
Compounds with an IC50_Mean < 100 uM are considered to be active in this assay.
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 (not utilized in this assay)
2) Second tier (41-80 range) is reserved for dose-response confirmation data for positives identified in the primary screening (not utilized in this assay)
3) Third tier (81-100 range) is reserved for dry-powder compounds
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 the IAP 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*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. Any compounds with the calculated score exceeding 100 are assigned the Score = 100. This results in the Score values in the range 85 to 100 for compounds that demonstrate high potency and predictable behavior. Compounds that are less active in the assay or whose concentration-dependent behavior are likely to be an artifact of the assay will generally have lower Score values.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)