uHTS absorbance assay for the identification of compounds that inhibit PHOSPHO1
Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of HA crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles more ..
BioActive Compounds: 3164
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084086-01
Assay Provider: Dr. Jose Luis Milan, Sanford-Burnham Medical Research Institute, San Diego CA
Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of HA crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles (MVs). Dr. Milan's working model is that bone mineralization is first initiated within the lumen of MVs. In a second step, HA crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu where they continue to propagate along collagen fibrils. Recent data have indicated that tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role in restricting the concentration of extracellular inorganic pyrophosphate (PP), a mineralization inhibitor, to maintain a P/PPi ratio permissive for normal bone mineralization. Using a variety of single and double gene knockout experiments it has been found that mice deficient in TNAP function, i.e., Akp2-/- mice, display osteomalacia due to an arrest in the propagation of HA crystals outside the MVs caused by an increase in extracellular PPi concentrations. Inside the MVs, however, HA crystals are still present in Akp2-/- mice. It is hypothesize that a newly identified soluble phosphatase, PHOSPHO1, with specificity for phosphoethanolamine (PEA) and phosphoserine (PS) present in the MVs, is responsible for increasing the local concentration of Pi inside the MVs to change the P/PPi ratio to favor precipitation of HA seed crystals.
This biochemical assay employs a colorimetric readout based on the enzyme's ability to liberate phosphate from phosphoethanolamine and its reaction with Biomol Green reagent.
1) POSPHO1 was obtained from the assay provider's laboratory.
2) Assay Buffer: 20mM MES-NaOH pH 6.7, 2mM MgCl2, 0.0125% Tween-20, 0.01% BSA
1) Dispense 1.5ul of assay buffer into columns 1 and 2 of a black, clear bottom Corning (#3891) 1536 well assay plate.
2) Dispense 1.5ul of PHOSPHO1 (2.5ng/ml) in assay buffer to wells in columns 3 through 48.
3) Using a HighRes biosolutions pintool dispense 20nl of 2mM compounds in DMSO to columns 5-48
4) Dispense 20nl of DMSO to columns 1-4.
5) Add 1.5ul of assay buffer containing 900uM phosphoethanolamine to all wells
6) Incubate lidded plate for 1 hour at room temp.
7) Add 3ul of Biomol
8) Incubate for 30 min to allow Biomol signal to develop
9) Read plate on a Perkin Elmer Viewlux at 630nm in absorbance mode
Ex1:2 = 630 DF10
Light energy = 100000
Measurement time = 5 sec
Compounds that demonstrated an inhibition of >= 60% at 13.3uM concentration are defined as actives 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 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 % displacement 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 and is not applicable to this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)