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BioAssay: AID 2074

SAR Fluorescence Assay for VHR1 Inhibitors using DiFMUP

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(9)
 
 
Active(9)
 
 
 Tested Substances
 Tested Substances
All(9)
 
 
Active(9)
 
 
AID: 2074
Data Source: Burnham Center for Chemical Genomics (BCCG-A251-VHR1-SAR-FluorescenceAssay-Using DiFMUP)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-28
Modify Date: 2011-01-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 9
Related Experiments
Show more
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
1992HTS Colorimetric assay for the identification of compounds that inhibit VHR1Screening depositor-specified cross reference
2004SAR Colorimetric assay for the identification of compounds that inhibit VHR1Confirmatory depositor-specified cross reference
1055In Vitro MKP-3 Dose Response Assay for SAR StudyConfirmatory same project related to Summary assay
1957SAR VHR1 Fluorescent Assay for In Vitro dose response studiesConfirmatory same project related to Summary assay
1958SAR VHR1 absorbance Assay for In Vitro dose response studies.Confirmatory same project related to Summary assay
2070MOA VHR1 Fluorescent secondary assay for identification of redox-state modulating compoundsConfirmatory same project related to Summary assay
2082SAR Fluorescence HePTP Assay for Selectivity Study of VHR1 Inhibitors using DiFMUPConfirmatory same project related to Summary assay
2083SAR Fluorescence MKP-1 Assay for Selectivity Study of VHR1 Inhibitors using DiFMUPConfirmatory 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 fluorescent readout based on the enzyme's ability to catalyze the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in the presence of an inhibitor.
The assay is developed and performed to study the structure-activity relationship on analogs of the confirmed hits reported in AID 2004. Compounds are either acquired from commercial sources or synthesized internally.

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
The VHR-catalyzed hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in the presence of compound was assayed at 30 degree celsius in a 60 ul 96-well format reaction system in 150 mM Bis-Tris, pH 6.0 assay buffer having an ionic strength of 150 mM (adjusted with NaCl) and containing 1 mM DTT and 5% DMSO. With a VHR concentration of 1 nM, and at various concentrations of the compound (between 0.1 nM and 10 uM), the initial rate at 78 uM DiFMUP concentration (Km value) was determined using a FLx800 micro plate reader (Bio-Tek Instruments, Inc.), an excitation wave length of 360 nm and measuring the emission of the fluorescent reaction product 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) at 460 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. The IC50 value was determined by plotting the relative activity versus inhibitor concentration and fitting to Equation 1 using the software GraphPad Prism (GraphPad Software, Inc.).
Vi/V0 = IC50/(IC50 + [I]) (Eq. 1)
In this case, Vi is the reaction velocity when the inhibitor concentration is [I], V0 is the reaction velocity with no inhibitor, and IC50 = Ki + Ki[S]/Km.
Comment
A positive of the assay is defined as a compound with IC50 greater than 50 uM.

Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration compound efficacy, the screening stage of the data and apparent compound behavior in the assay.

The the scoring system is as follows:
1) First tier (0-40 range) is reserved for primary screening data and therefore is not applicable in this assay.

2) Second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits and therefore is not applicable in this assay.

3) Third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues and utilized for SAR studies.
a. Compounds that failed to reproduce from dry powder or have IC50 > 50 uM are assigned inactive and a score value of 81.
b. The score is linearly correlated with a compound's potency using the following equation:
Score = 82+3*(pIC50-4)
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HeLa
From ChEMBL:
Assay Type: Binding
Result Definitions
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 "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50*IC50 value determined using Vi/V0 = IC50/(IC50 + [I])FloatμM

* Activity Concentration.
Additional Information
Grant Number: MH084230-01A1

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