HTS identification of compounds activating TNAP at intermediate concentration of phosphate acceptor detected in 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 human, 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: 8
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: 1R03 MH082385-01
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 human, 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.
The goal of this HTS is to identify novel and specific activators of TNAP. 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 DEA. Therefore, for detection of compounds with diverse mode of action, the HTS assay was optimized and HTS campaigns were performed in the presence and in the absence of DEA. The current AID reports a set of the data from screening in the presence of intermediate concentration of DEA.
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). RO3 submission, MH082385-01: Activators of the Pyrophosphatase Activity of Alkaline Phosphatase. Assay Providers: Drs. Jose Luis Millan and Eduard Sergienko, Sanford-Burnham Medical Research Institute, San Diego, CA.
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: 250 mM DEA, pH 9.8, 2.5 mM MgCl2, and 0.05 mM ZnCl2.
3)TNAP working solution contained a 1/800 dilution in assay buffer.
4)CDP-star working solution contained 125 uM CDP-star in MQ water.
5)Negative Control (NC) solution - 5 mM levamisole in 10% DMSO.
6)Positive Control (PC) solution - 10% DMSO.
TNAP HTS protocol:
1) 4 uL of PC solution was added to column 1 using the Thermo wellmate dispenser
2) 4 uL of NC solution was added to column 2 using the Thermo wellmate dispenser
3) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well white small volume plates (784075) using BioMek FX.
4) 8 uL of TNAP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
5) 8 uL of CDP-star working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
6) 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. 1/2000 dilution TNAP (columns 1-24)
c. 50 uM CDP-star (columns 1-24)
d. 1 mM Levamisole (columns 2)
e. 2 % DMSO (columns 1-24)
f. 20 uM compounds (columns 3-24)
7) Plates were incubated for 30 mins at room temperature.
8) Luminescence was measured on the Envision plate reader (Perkin Elmer).
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
Data Table (Concise)