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BioAssay: AID 1992

HTS Colorimetric 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 ..
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 Tested Compounds
 Tested Compounds
All(49812)
 
 
Active(347)
 
 
Inactive(49465)
 
 
 Tested Substances
 Tested Substances
All(49840)
 
 
Active(347)
 
 
Inactive(49493)
 
 
AID: 1992
Data Source: Burnham Center for Chemical Genomics (BCCG-A231-VHR1-Colorimetric-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-19
Modify Date: 2010-12-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 347
Related Experiments
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AIDNameTypeProbeComment
1654uHTS absorbance assay for the identification of compounds that inhibit VHR1.Confirmatory depositor-specified cross reference
1661Summary of the absorbance assay for the identification of compounds that inhibit VHR1.Summary1 depositor-specified cross reference
1878Fluorescent assay for identification of compounds that inhibit VHR1Confirmatory depositor-specified cross reference
1957SAR VHR1 Fluorescent Assay for In Vitro dose response studiesConfirmatory depositor-specified cross reference
1958SAR VHR1 absorbance Assay for In Vitro dose response studies.Confirmatory depositor-specified cross reference
2004SAR Colorimetric assay for the identification of compounds that inhibit VHR1Confirmatory depositor-specified cross reference
2074SAR Fluorescence Assay for VHR1 Inhibitors using DiFMUPConfirmatory depositor-specified cross reference
2082SAR Fluorescence HePTP Assay for Selectivity Study of VHR1 Inhibitors using DiFMUPConfirmatory depositor-specified cross reference
2083SAR Fluorescence MKP-1 Assay for Selectivity Study of VHR1 Inhibitors using DiFMUPConfirmatory depositor-specified cross reference
1055In Vitro MKP-3 Dose Response Assay for SAR StudyConfirmatory same project related to Summary assay
2070MOA VHR1 Fluorescent secondary assay for identification of redox-state modulating compoundsConfirmatory same project related to Summary assay
2678SAR analysis of chemical inhibitors of HePTP using a Fluorescent assay - Set 2Confirmatory same project related to Summary assay
2679SAR analysis of inhibitors of MKP-3 - Set 2Confirmatory same project related to Summary assay
2684SAR VHR1 Fluorescent Assay for In Vitro dose response studies Set 2Confirmatory same project related to Summary assay
449733SAR analysis of compounds that inhibit VHR1, Fluorescent Assay - Set 2Confirmatory same project related to Summary assay
488861Dose Response Confirmation of compounds that inhibit VHR1 in Fluorescent AssayConfirmatory same project related to Summary assay
488923Dose Response confirmation of compounds that inhibit HePTPConfirmatory same project related to Summary assay
540288SAR VHR1 Fluorescent Assay for In Vitro dose response studies Set 3Confirmatory same project related to Summary assay
Description:
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 p-nitrophenyl phosphate (pNPP) and its reaction with Biomol Green reagent.

References:
Wu, S., Vossius, S., Rahmouni, S., Miletic, A.V., Vang, T., Vazquez-Rodriguez, J., Cerignoli, F., Arimura, Y., Williams, S., Hayes, T., Vasile, S., Pellecchia, M., Mustelin, T., and Tautz, L. (2009) Multidentate Small-Molecule Inhibitors of Vaccinia H1-Related (VHR) Phosphatase Decrease Proliferation of Cervix Cancer Cells. J. Med. Chem., Article ASAP (http://dx.doi.org/10.1021/jm901016k)

Henkens, R., Delvenne, P., Arafa, M., Moutschen, A., Zeddou, M., Tautz, L., Boniver, J., Mustelin, T., and Rahmouni, S. (2008) Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity proteinphosphatase VHR. BCM Cancer 8:147.

Tautz L. and Mustelin T. (2007) Strategies for Developing Protein Tyrosine Phosphatase Inhibitors. Methods 42:250-60.
Protocol
Chemical Library Screening. A set of 50,000 drug-like molecules from the DIVERSet library (ChemBridge, Inc.) was screened in a 96-well format in vitro phosphatase assay. Compounds had a working concentration of 0.15 mg/ml in 10% DMSO. Each reaction contained 180 nM VHR, 4 mM pNPP, and 0.02 mg/mL compound in 0.1 M Bis-Tris pH 6.0 reaction buffer with 1 mM dithiothreitol (DTT) present. The final volume amounted to 70 uL and contained 1.4% DMSO. The reaction was initiated by addition of pNPP after a preincubation of the enzyme with compounds for 10 min at room temperature. After 15 min, the reaction was quenched by addition of 140 uL BIOMOL GREEN Reagent, and the pNPP hydrolysis was determined by measuring the absorbance of the complexed free phosphate at 620 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the negative control without addition of enzyme. Other controls included a positive control (no inhibitor added), a background control (no substrate added), and a control with 200 uM of the general PTP inhibitor sodium orthovanadate. To quantitate the inhibitory efficacy of the library compounds, we determined the ratio of inhibition in comparison to the positive control. Every compound with >=60% inhibition was cherry-picked and rescreened using the same assay format. Compounds that showed >=60% inhibition also in the confirmatory screen were regarded as hit. A total of 221 hits were identified in the confirmation screen.
Comment
Compounds that demonstrated an inhibition of >= 60% at 20uM concentration are defined as actives of primary HTS assay. These compounds were retested in a single concentration confirmation screen. Compounds with >= 60% inhibition in the reconfirmation assay are considered active.
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
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 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 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HeLa
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%Inhibition of Repeat at 20uM (20μM**)Average of the % inhibition of the cherry picked primary hits retested at 20 uMFloat%
6%Inhibition at 20uM (20μM**)% Inhibition in primary screeningFloat%
7Mean HighMean Absorbance of negative controls in the corresponding plateFloatOD
8STD Deviation HighMean Absorbance of negative controls in the corresponding plateFloatOD
9Mean LowMean Absorbance of positive controls in the corresponding plateFloatOD
10STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatOD

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084230-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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