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BioAssay: AID 1077

Fluorescent secondary assay for dose-response confirmation of chemical inhibitors of HePTP

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 ..
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 Tested Compounds
 Tested Compounds
All(241)
 
 
Active(146)
 
 
Inactive(96)
 
 
 Tested Substances
 Tested Substances
All(245)
 
 
Active(149)
 
 
Inactive(96)
 
 
AID: 1077
Data Source: Burnham Center for Chemical Genomics (SDCCG-A052-HePTP (Fluorescent 2nd assay))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-03-03
Modify Date: 2010-09-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 146
Related Experiments
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AIDNameTypeProbeComment
521HTS Discovery of Chemical Inhibitors of HePTP, a Leukemia TargetConfirmatory depositor-specified cross reference
1059In Vitro HePTP Dose Response Colorimetric Assay for SAR StudyConfirmatory depositor-specified cross reference
1784Summary of small molecule inhibitors of LYP via a fluorescence intensity assaySummary depositor-specified cross reference
2068MOA HePTP Fluorescent secondary assay for identification of redox-state modulating compoundsConfirmatory depositor-specified cross reference
2085Summary assay for inhibitors of HePTPSummary2 depositor-specified cross reference
2134HTS HePTP Fluorescent Assay using OMFP substrate for In Vitro dose response studiesConfirmatory depositor-specified cross reference
2678SAR analysis of chemical inhibitors of HePTP using a Fluorescent assay - Set 2Confirmatory depositor-specified cross reference
435032Dose Response confirmation of HTS hits from an HePTP Fluorescent Assay using OMFP substrate - Set 2Confirmatory depositor-specified cross reference
449736SAR analysis of compounds that inhibit HePTP - Set 2Confirmatory depositor-specified cross reference
1779uHTS identification of small molecule inhibitors of LYP via a fluorescence intensity assayConfirmatory same project related to Summary assay
2126LYP1 Fluorescent Assay using OMFP substrate for In Vitro dose response studies.Confirmatory same project related to Summary assay
2135SAR LYP1 Fluorescent Assay using pCAP substrate for In Vitro dose response studiesConfirmatory same project related to Summary assay
2140SAR LYP1 Fluorescent Assay using OMFP substrate for In Vitro dose response studiesConfirmatory same project related to Summary assay
2682SAR LYP1 Fluorescent Assay using pCAP substrate for In Vitro dose response studies - Set 2Confirmatory same project related to Summary assay
2686SAR LYP1 Fluorescent Assay using OMFP substrate for In Vitro dose response studies - Set 2Confirmatory same project related to Summary assay
435024Dose Response confirmation of uHTS hits from a small molecule inhibitors of LYP via a fluorescence intensity assay - Set 2Confirmatory same project related to Summary assay
435027Dose Response confirmation of uHTS hits from a small molecule inhibitors of LYP via a fluorescence intensity assay using pCAP substrateConfirmatory same project related to Summary assay
449726SAR LYP1 Fluorescent Assay using OMFP substrate for In Vitro dose response studies - Set 3Confirmatory same project related to Summary assay
449727SAR LYP1 Fluorescent Assay using pCAP substrate for In Vitro dose response studies - Set 3Confirmatory same project related to Summary assay
488869SAR LYP1 Fluorescent Assay using OMFP substrate for In Vitro dose response studies - Set 4Confirmatory same project related to Summary assay
488871SAR LYP1 Fluorescent Assay using pCAP substrate for In Vitro dose response studies - Set 4Confirmatory same project related to Summary assay
488884SAR Selectivity Analysis of small molecule inhibitors of PEST using pCAP in a fluorescence assayConfirmatory same project related to Summary assay
488889SAR analysis of compounds that inhibit HePTP - Set 3Confirmatory same project related to Summary assay
1055In Vitro MKP-3 Dose Response Assay for SAR StudyConfirmatory same project related to Summary assay
1957SAR VHR1 Fluorescent Assay for In Vitro dose response studiesConfirmatory same project related to Summary assay
488889SAR analysis of compounds that inhibit HePTP - Set 3Confirmatory same project related to Summary assay
488923Dose Response confirmation of compounds that inhibit HePTPConfirmatory same project related to Summary assay
Description:
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.
Protocol
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
Comment
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
4IC50_1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
8IC50_2IC50 value determined using sigmoidal dose response equationFloatμM
9Std.Err(IC50)_2Standard Error of IC50 valueFloatμM
10nH_2Hill coefficient determined using sigmoidal dose response equationFloat
11IC50_Qualifier_3This qualifier is to be used with the next TID, IC50_3. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
12IC50_3IC50 value determined using sigmoidal dose response equationFloatμM
13Std.Err(IC50)_3Standard Error of IC50 valueFloatμM
14nH_3Hill coefficient determined using sigmoidal dose response equationFloat
15IC50_Qualifier_4This qualifier is to be used with the next TID, IC50_4. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
16IC50_4IC50 value determined using sigmoidal dose response equationFloatμM
17Std.Err(IC50)_4Standard Error of IC50 valueFloatμM
18nH_4Hill coefficient determined using sigmoidal dose response equationFloat
19IC50_Qualifier_5This qualifier is to be used with the next TID, IC50_5. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
20IC50_5IC50 value determined using sigmoidal dose response equationFloatμM
21Std.Err(IC50)_5Standard Error of IC50 valueFloatμM
22nH_5Hill coefficient determined using sigmoidal dose response equationFloat
23IC50_Qualifier_6This qualifier is to be used with the next TID, IC50_6. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
24IC50_6IC50 value determined using sigmoidal dose response equationFloatμM
25Std.Err(IC50)_6Standard Error of IC50 valueFloatμM
26nH_6Hill coefficient determined using sigmoidal dose response equationFloat
27IC50_Qualifier_7This qualifier is to be used with the next TID, IC50_7. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
28IC50_7IC50 value determined using sigmoidal dose response equationFloatμM
29Std.Err(IC50)_7Standard Error of IC50 valueFloatμM
30nH_7Hill coefficient determined using sigmoidal dose response equationFloat

* Activity Concentration.
Additional Information
Grant Number: XO1 MH077603-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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