uHTS identification of compounds activating TNAP at a high concentration of phosphate acceptor detected in a luminescent assay
Alkaline phosphatases (EC 126.96.36.199) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organisms. In humans, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-non specific, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there is therapeutic potential of modulating TNAP activity. ..more
BioActive Compounds: 5
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: R03 MH082385-01
Assay Provider: Dr. Jose Luis Millan, Sanford-Burnham Medical Research Institute, San Diego, CA
Alkaline phosphatases (EC 188.8.131.52) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in the most organisms. In humans, four isozymes of APs have been identified. Three isozymes are tissue-specific and the fourth one is tissue-non specific, named TNAP. TNAP deficiency is associated with defective bone mineralization in the form of rickets and osteomalacia. Therefore, there is therapeutic potential of modulating TNAP activity.
TNAP luminescent assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG), part of the Molecular Library Screening Center Network (MLSCN). The goal of this HTS is to identify activators of TNAP with a novel mode of action. The only known to date class of alkaline phosphatases activators are nitrogen-containing alcohols, such as diethanolamine (DEA), that act as a phosphoacceptor substrate and exhibit its effect in high-mM concentration range. Identification of potent compounds mimicking the effect of the phosphoacceptors was addressed in a previous screening campaign (AID 1001). The current assay was specifically optimized and performed for identification of compounds with a different mechanism of action. To this end, we utilized a saturating concentration of DEA of 1 M in the assay: compounds with a mode of action similar to DEA are expected to demonstrate diminished stimulating potential if tested in the presence of DEA.
TNAP assay materials:
1) TNAP protein was provided by Dr. Jose Luis Millan (Sanford-Burnham Medical Research Institute, San Diego, CA). The CDP-star was obtained from New England Biolabs.
2) Assay Buffer: 2 M DEA, pH 9.8, 2 mM MgCl2, and 0.04 mM ZnCl2.
3) TNAP working solution contained a 1/400 dilution in assay buffer.
4) CDP-star working solution contained 200 uM CDP-star in MQ water.
5) Negative Control (NC) solution: 500 mM EDTA
6) Positive Control (PC) solution: 100% DMSO
TNAP HTS protocol:
1) 2 uL of CDP-star working solution was added to all the well of Costar 1536-well white plate (cat #3725) using a Thermo Multidrop Combi dispenser
2) 40 nL of NC solution was added to columns 1-2 using a HighRes biosolutions pintool and V&P Scientific pins
3) 40 nL of PC solution was added to columns 3-4 using a HighRes biosolutions pintool and V&P Scientific pins
3) 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5-48 a HighRes biosolutions pintool and V&P Scientific pins
4) 2 uL of TNAP working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
5) Final concentrations of the components in the assay were as follows:
a. 1 M DEA, pH 9.8, 1.0 mM MgCl2, 0.02 mM ZnCl2
b. 1/800 dilution TNAP
c. 100 uM CDP-star
d. 20 uM compounds
6) Plates were incubated for 30 mins at room temperature.
7) Luminescence was measured on a ViewLux plate reader (Perkin Elmer).
8) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
9) Data analysis was performed using CBIS software (ChemInnovations, Inc).
TNAP activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC),
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 TNAP 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 TNAP 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 TNAP assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and the score is correlated with TNAP activation factor demonstrated by a compound at 20 uM concentration:
a. If AF<1, then the assigned score is 0
b. For all other AF values,
Score = 40 - 40/AF
This formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
** Test Concentration.
Data Table (Concise)