HTS Discovery of Chemical Inhibitors of HePTP, a Leukemia Target
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 more ..
BioActive Compounds: 724
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 colorimetric HTS assay was developed and run to identify HePTP inhibitors.
HePTP assay materials:
1) HePTP protein was provided by Dr. Mustelin (Sanford-Burnham Medical Research Institute, San Diego, CA). The pNPP pellets were obtained from Sigma-Aldrich (S0942). Biomol Green reagent was purchased from BIOMOL Research Laboratories, Inc (AK-111)
2) Assay Buffer: 50 mM Bis-Tris, pH 6.0, 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) pNPP working solution contained 1 mM pNPP in MQ water. Solution was prepared fresh prior to use.
5) Vanadate working solution - 45 mM Na3VO4 in 10% DMSO
HePTP HTS protocol:
1) 4 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well clear microtiter plates (781101).
2) 4 uL of the following solutions were using the Multidrop bulk dispenser (Thermo):
a. 10% DMSO column 1 (negative control)
b. 45 mM Na3VO4 in 10% DMSO - column 2 (positive control).
3) 8 uL of HePTP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4) 8 uL of pNPP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
5) Final concentrations of the components in the assay were as follows:
a. 20 mM Bis-Tris, pH 6.0, 1.0 mM DTT, 0.005% Tween 20.
b. 2.75 nM HePTP (columns 1-24)
c. 0.4 mM pNPP (columns 1-24)
d. 9 mM Na3VO4 (column 2)
e. 2 % DMSO (columns 1-24)
f. 20 uM compounds (columns 3-24)
6) Plates were incubated for 1h at room temperature.
7) 40 uL of Biomol Green reagent was added to the entire plate using the WellMate bulk dispenser (Matrix).
8) Plates were incubated for 30 mins at room temperature
9) Absorbance at 620 nM was measured on the Envision plate reader (PerkinElmer).
10) Data analysis was performed using CBIS software (ChemInnovations, Inc).
HePTP dose-response confirmation screening 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 pNPP working solution was added to the whole plate using WellMate bulk dispenser (Matrix).
4) Plates were incubated for 1h at room temperature.
5) Absorbance at 620 nM was measured on the Envision plate reader (PerkinElmer).
6) Data analysis was performed using CBIS software (ChemInnovations, Inc) using sigmoidal dose-response equation through non-linear regression
This assay was screened through two compound library collections. The first library was the NIH collection, which is supplied to all of the Molecular Libraries Screening Centers. The second library consisted of 50,000 compounds from ChemBridge Corp. The compounds in the ChemBridge collection were screened in mg/ml, which is different from the NIH library which was screened in mM. To designate this difference the comment line in the results table will state the following 'Primary testing at 3.333 ug/ml' for data from the Chembridge set.
Compounds with greater than 50% inhibition of HePTP at testing concentration are defined as actives of the primary screening. The primary screening actives proceed to the dose-response confirmation stage. Compounds that demonstrate IC50 values in the range of analyzed concentrations remain active in the outcome column. Compounds that failed dose-response confirmation are downgraded to inactive in the outcome field.
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 HePTP assay is described below.
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 HePTP assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data the score is correlated with % displacement 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
a.Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b.The score is linearly correlated with a compound's potency using the following equation
Score = 44 + 6*(pIC50-3),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate IC50> 100 uM.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)