Single concentration confirmation of uHTS hits from a small molecule inhibitors of mouse intestinal alkaline phosphatase via a luminescent assay
Alkaline phosphatase (EC 184.108.40.206) (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: 509
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 220.127.116.11) (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.
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
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
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 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
** Test Concentration.
Data Table (Concise)