SAR Fluorescence HePTP Assay for Selectivity Study of 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 ..
BioActive Compounds: 5
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.
Hematopoietic tyrosine phosphatase (HePTP) is a classical pTyr-specific phosphatase. HePTP shares a physiological substrate with VHR, namely the extracellular signal-regulated kinases Erk1/2, and therefore was chosen to assess the selectivity of the VHR1 inhibitors reported in AID 2004.
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.
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.
The HePTP-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 HePTP concentration of 1 nM, and at various concentrations of the compound (between 1 nM and 100 uM), the initial rate at 14 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.
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 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
* Activity Concentration.
Data Table (Concise)