Luminescent assay for identification of inhibitors of bovine intestinal 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 ..
Depositor Specified Assays
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: 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.
IAP is inhibited by a number of inhibitors. They include L-phenylalanine, L-tryptophan, L-leucine and phenylalanine-glycylglycine. 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 for IAP. Bovine enzyme was utilized in this primary screening as a highly active alternative with high homology to the human enzyme.
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)Bovine IAP protein and CDP-star substrate were obtained from Biozyme Laboratories and Applied Biosystems, respectively.
2)Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl2.
3)IAP working solution contained a 1/26,000,000 dilution in assay buffer.
4)CDP-star working solution contained 75 uM CDP-star in MQ water.
IAP primary HTS protocol:
1)4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well white small volume plates (784075).
2)Using the Thermo WellMate the following solutions were added:
a.4 uL of 10% DMSO were added to columns 1-2
b.8 uL of water was added to column 1 (negative control)
c.8 uL of IAP working solution was added to column 1-24
d.8 uL of CPD-star was added to columns 2-24
3)Final concentrations of the components in the assay were as follows:
a.100 mM DEA, pH 9.8, 1.0 mM MgCl2, 0.02 mM ZnCl2 (columns 1-24)
b.1/65,000,000 dilution IAP (columns 1-24)
c.30 uM CDP-star (columns 2-24)
d.2% DMSO (columns 1-24)
e.20 uM compounds (columns 3-24)
4)Plates were incubated for 30 mins at room temperature.
5)Luminescence was measured on the Envision plate reader (Perkin Elmer).
6)Data analysis was performed using CBIS software (ChemInnovations, Inc).
Compounds with greater than 50% inhibition of IAP at 20 uM concentration are defined as actives of the primary screening.
Activity scoring rules for IAP HTS are as follows:
1) First tier (0-40 score range) is reserved for primary screening data - the score is correlated with % inhibition in the assay demonstrated by a compound at 20 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
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 (not utilized in this assay)
** Test Concentration.
Data Table (Concise)