Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Number: XO1 MH077603-01
Assay Provider: Dr. Tomas Mustelin, Sanford-Burnham Medical Research Institute


Protein tyrosine phosphatases (PTPs), working with protein tyrosine kinases (PTKs), control the phosphorylation state of many proteins in the signal transduction pathways. HePTP is a tyrosine phosphatase expressed in hematopoietic cells and regulates the MAP kinases Erk and p38. It has been found that HePTP is often dysregualted in the preleukemic disorder myelodysplastic syndrome, as well as in acute myelogeneous leukemia. Small molecule inhibitors of HePTP will be useful as molecular probes for studying the mechanism of signal transduction and MAP kinase regulation, and may have therapeutic potential for the treatment of hematopoietic malignancies.

This fluorescent assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics as a secondary assay for side-by-side study with the compounds tested in the "In Vitro HePTP Dose Response Colorimetric Assay for SAR Study" assay, AID 1059) Enzyme activity and its inhibition by screened compounds was measured in a kinetic assay based on hydrolysis of 3-O-methylfluorescein phosphate (OMFP) resulting in a fluorescence increase due to 3-O-methylfluorescein release.

HePTP assay materials:
1)HePTP protein was provided by Dr. Mustelin (Sanford-Burnham Medical Research Institute, San Diego, CA).
2)Assay Buffer: 50 mM Bis-Tris, pH 6.0, 375 mM NaCl, 2.5 mM DTT, 0.0125% Tween 20.
3)HePTP working solution contained 6.875 nM HePTP in assay buffer. Solution was prepared fresh prior to use.
4)The OMFP 20 mM stock solution was prepared by dissolving 10.5 mg OMFP in 1 mL DMSO and sonicating the solution for 1 min. Right before use, the stock solution was diluted to a working solution of 750 uM concentration.
5)Vanadate working solution - 45 mM Na3VO4 in 10% DMSO


HePTP dose-response assay protocol:
1)Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well white small-volume plates (784075). Columns 1-2 and 23-24 contained 4 uL of vanadate working solution and 10% DMSO, respectively.
2)8 uL of HePTP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
3)8 uL of OMFP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4)Fluorescence (ex/em: 485/525 nm, 515 nm cutoff) was measured on M5 plate reader (Molecular Devices).
5)The initial slope of the 4-min progress curve was obtained using SoftMax Pro software (Molecular Devices).
6)Data analysis was performed using CBIS software (ChemInnovations, Inc) using sigmoidal dose-response equation through non-linear regression

A positive of the assay is defined as a compound with IC50 value in the range of tested concentrations, i.e. below 100 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. Details of the Scoring System will be published elsewhere.

Briefly, the outline of the scoring system utilized for the HePTP assay 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 > 100 uM are assigned inactive and a score value of 81.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of a target- or a pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on the expectation that a compound with a single mode of action that achieved an equilibrium in the assay would demonstrate the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their divergence.
c. The score is calculated using the following equation:
Score = 82+3*(pIC50-4)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units, and QC is calculated using Hill coefficient as above. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior in the assays.

Grant Number: XO1 MH077603-01