uHTS absorbance assay for the identification of compounds that inhibit VHR1.
Protein tyrosine phosphatases (PTPs) play vital roles in numerous cellular processes and are implicated in a growing number of human diseases, ranging from cancer to cardiovascular, immunological, infectious, neurological, and metabolic diseases. The Vaccinia H1-related (VHR) PTP is a dual-specific Erk and Jnk phosphatase, the loss of which causes specific cell cycle arrest in HeLa carcinoma more ..
BioActive Compounds: 1524
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 Number: 1 R03 MH084230-01A1
Assay Provider: Dr. Lutz Tautz, Sanford-Burnham Medical Research Institute, San Diego CA
Protein tyrosine phosphatases (PTPs) play vital roles in numerous cellular processes and are implicated in a growing number of human diseases, ranging from cancer to cardiovascular, immunological, infectious, neurological, and metabolic diseases. The Vaccinia H1-related (VHR) PTP is a dual-specific Erk and Jnk phosphatase, the loss of which causes specific cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells without detrimental effects on normal cells. Recent studies by collaborators and us suggest that VHR is upregulated in several cervix cancer cell lines, in squamous intraepithelial lesions, and squamous cell carcinomas of the uterine cervix.
This biochemical assay employs a colorimetric readout based on the enzyme's ability to liberate phosphate from para-nitrophenylphosphate (PNPP) and its reaction with Biomol Green reagent.
Tautz L. and Mustelin T. 2007. Methods 42:250-60.
1) VHR1 was obtained from the assay provider's laboratory.
2) Assay Buffer: 20mM Bis-Tris pH 6.0, 1mM DTT, 0.005% Tween-20
3) Biomol Green - Biomol (Cat #AK111-1000)
1) Dispense 1.5ul of assay buffer into columns 1 and 2 of a black, clear bottom Corning (#3891) 1536 well assay plate.
2) Add 1.5ul of 1474uM pNPP in assay buffer to all wells
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) Dispense 1.5ul of 40nM VHR1 in assay buffer to wells in columns 3 through 48.
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
1) Dispense 10ul of assay buffer into columns 1 and 2 of Greiner 384-well clear plates (#781101).
2) Add 10ul of 250uM pNPP in water to all wells
3) Dispense serially diluted compounds to columns 3-22 and DMSO to columns 1-2 and 23-24. Final concentration DMSO in the assay is 1.575%
4) Dispense 10ul of 40nM VHR1 in assay buffer to wells in columns 3-24.
5) Incubate lidded plate for 1 hour at room temp.
6) Add 20uL of Biomol to all wells
7) Incubate for 30 min to allow Biomol signal to develop
8) Read plate on M5 plate reader in absorbance mode at 620 nm.
Compounds that demonstrated an inhibition of >= 50% at 13.3uM concentration are defined as actives of primary HTS assay.
These compounds were retested in triplicate in a single concentration confirmation screen. Compounds with an average of >= 50% inhibition in the reconfirmation assay are considered active.
The hits were then confirmed in dose-response assay. Compounds that demonstrate IC50 < 100 uM considered active in the confirmation 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 % inhibition in the assay demonstrated by a compound at 13.3 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
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the VHR1 assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)