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.

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.

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.

Grant Number: 1 R03 MH084230-01A1