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
Description. 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.

This dose response assay is developed and performed to confirm hits originally identified in "HTS Colorimetric assay for the identification of compounds that inhibit VHR1" (AID 1992) and to study the structure-activity relationship on analogs of the confirmed hits. 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.

Ki Determination. Ki values were determined for the top 56 compounds in AID 1992, "HTS Colorimetric assay for the identification of compounds that inhibit VHR1", averaging 90% inhibition or higher in both, primary and confirmatory screen. The PTP-catalyzed hydrolysis of pNPP in the presence of compound was assayed at 30 oC in a 100 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. The enzyme was preincubated with various fixed concentrations of inhibitors for 10 min. The reaction was initiated by addition of various concentrations of pNPP (ranging from 0.2 to 10 Km) to the reaction mixture. The initial rate was determined using an ELx808 micro plate reader (Bio-Tek Instruments, Inc.), reading the absorbance of the product p-nitrophenol at 404 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. The inhibition constant and inhibition pattern was evaluated by fitting the data to the Michaelis-Menten equations for either competitive, uncompetitive or mixed inhibition as described before (1), using nonlinear regression and the program GraphPad Prism (GraphPad Software, Inc.). For a comparison of the fitting results the second-order Akaike's Information Criterion (AICc) was calculated as previously described.

Reference:
1) Tautz, L.; Bruckner, S.; Sareth, S.; Alonso, A.; Bogetz, J.; Bottini, N.; Pellecchia, M.; Mustelin, T. Inhibition of Yersinia tyrosine phosphatase by furanyl salicylate compounds. J Biol Chem 2005, 280, 9400-9408.

Compounds with a Ki < 20 uM are considered to be active 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
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 and is not applicable to this assay.
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
a. Inactive compounds are assigned a score value equal to 81.
b. The score is linearly correlated with a compound potency. For active compounds:
Score = 82 + (20-ki)*0.9

Grant Number: 1 R03 MH084230-01A1