uHTS Luminescent assay for identification of activators of mouse intestinal alkaline phosphatase
Assay Provider: Dr. Jose Luis Millan, Sanford-Sanford-Burnham Medical Research Institute, San Diego, CA. ..more
BioActive Compounds: 785
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 (MLSPN)
Grant Proposal Number: X01-MH077602-01
Assay Provider: Dr. Jose Luis Millan, Sanford-Sanford-Burnham Medical Research Institute, San Diego, CA.
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: 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 HTS is to identify novel and specific activators of mouse IAP. 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.
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 muIAP 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
IAP activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_PC)/(Mean_NC - Mean_PC)
where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate.
Compounds with greater than or equal to 2-fold activation (AF >= 2) of IAP at 20-uM concentration are defined as actives of the primary screening.
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)