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BioAssay: AID 1055

In Vitro MKP-3 Dose Response Assay for SAR Study

This MKP-3 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the MKP-3 in vitro HTS assay (AID 425) ..more
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 Tested Compounds
 Tested Compounds
All(189)
 
 
Active(62)
 
 
Inactive(128)
 
 
 Tested Substances
 Tested Substances
All(193)
 
 
Active(64)
 
 
Inactive(129)
 
 
AID: 1055
Data Source: Burnham Center for Chemical Genomics (SDCCG-A047-MKP-3 (in vitro SAR))
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-02-28
Modify Date: 2011-07-27

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 62
Depositor Specified Assays
AIDNameTypeProbeComment
566In vitro MKP-3 Phosphatase Dose Response Active/Probe Assessment Assay - Reproducibility testingconfirmatory
553In vitro MKP-3 Phosphatase Dose Response Hit/Probe Assessment Assayconfirmatory
425MKP-3 in vitro HTS assayconfirmatory
1661Summary of the absorbance assay for the identification of compounds that inhibit VHR1.summary1 Summary AID.
1052MKP-3 in vitro secondary assay for identification of irreversible and slow-binding inhibitorsconfirmatory
2085Summary assay for inhibitors of HePTPsummary2
2679SAR analysis of inhibitors of MKP-3 - Set 2confirmatory
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 MH076390-01
Assay Provider: Dr. John Lazo, University of Pittsburg


This MKP-3 dose response assay is developed and performed for the purpose of SAR study on analogs of hits originally identified in the MKP-3 in vitro HTS assay (AID 425)

MKP-3 (mitogen-activated protein kinase phosphatase-3; EC 3.1.3.48, EC 3.1.3.16), a dual specificity phosphatase negatively regulates ERK1/2 by catalyzing the removal of a phosphoryl group from Thr(P) and Tyr(P) in the activation loop consensus motif -pTXpY.

MKP-3 screening was performed using a biochemical assay developed at the laboratory of Prof. John Lazo (University of Pittsburg). The assay was optimized and run at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN).

Enzyme activity and its inhibition by screened compounds was measured in an end-point assay based on hydrolysis of 3-O-methylfluorescein phosphate (OMFP) resulting in a fluorescence increase due to 3-O-methylfluorescein release.
Protocol
Materials
1) MKP-3 protein:
MKP-3 plasmid was obtained from Prof. John Lazo (University of Pittsburg). MKP-3 recombinant protein was expressed at the Sanford-Burnham Institute Protein Expression and Purification Facility according to the following protocol. Overnight culture of BL21 cells containing pET 21a - 6-His-MKP3 was grown in LB/amp overnight at 37 degrees Celsius; 100ml of the culture was added to 700 mL LB/amp and continued growing at 37 degrees Celsius until OD600 = 0.6-1.0. After induction with 1 mM IPTG, the culture was transferred to 28 degrees Celsius and grown for 3h. Harvested cells were resuspended in 30 mM Tris-HCl, pH 7.9, containing 500 mM NaCl, 2 mM DTT, 10% glycerol (binding buffer) and frozen in liquid nitrogen. Cells were resuspended in 25 mL binding buffer supplemented with 4 mM DTT and the protease inhibitors, and lysed in cell a homogenizer. The supernatant was loaded on a 2-mL Ni-NTA column and, after extensive column washing; MKP-3 was eluted with 200 mM imidazole, 30 mM Tris-HCl, pH 7.9, containing 100 mM NaCl, 2 mM DTT, 10% glycerol. Fractions containing MKP-3 were pooled and dialyzed in 30mM Tris-HCl, pH 7.0, 100mM NaCl, 2mM DTT, and 10% glycerol. The protein was stored at -80 degrees Celsius.
2) Assay Buffer (2.5X): 75 mM Tris, pH 7.0, 62.5 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 0.0125% Tween 20.
3) The MKP-3 working solution contained 2.5 ug/ml MKP-3 in 2.5X assay buffer.
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 125 uM concentration.

MKP-3 primary screening protocol
1) 5 uL of 100 uM compounds in 10% DMSO were dispensed in columns 3-24 of Greiner 384-well black plates (781076). Column 1 contained 5 uL of 10% DMSO (negative control). Column 2 contained 5 uL of 45 mM sodium orthovanadate in 10% DMSO.
2) 10 uL of the MKP-3 working solution was added to the whole plate using a WellMate bulk dispenser (Matrix Technologies, Inc).
3) 10 uL of the OMFP working solution was added to the whole plate using a WellMate bulk dispenser (Matrix Technologies, Inc).
4) Final concentrations of the components in the assay were as follows:
a. 30 mM Tris-HCl, pH 7.0, 25 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.005% Tween 20.
b. 1 ug/ml MKP-3
c. 50 uM OMFP
d. 2 % DMSO
e. 9 mM sodium orthovanadate (column 2)
f. 20 uM compounds (columns 3-24)
5) The reaction was allowed to progress for 70 min at room temperature.
6) The reaction was stopped by adding 50 uL of 100 mM NaOH.
7) Fluorescence intensity was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter - 530 nM, dichroic mirror - 505 nM. The signal for each well was acquired for 10 ms at medium attenuator.
8) Data analysis was performed using CBIS software (ChemInnovation, Inc).

MKP-3 secondary screening protocol
1) Dose-response curves contained 10 concentrations of compounds obtained using a 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 5uL compound in 10% DMSO were transferred into columns 3-22 of Greiner 384-well black small-volume plates (784076). Columns 1-2 contained 5 uL of 45 mM sodium orthovanadate in 10% DMSO. Columns 23-24 contained 5 uL of 10% DMSO (negative control).
2) 10 uL of the MKP-3 working solution was added to the whole plate using a WellMate bulk dispenser (Matrix Technologies, Inc).
3) 10 uL of the OMFP working solution was added to the whole plate using a WellMate bulk dispenser (Matrix Technologies, Inc).
4) The reaction progress was monitored in plate kinetic mode on an M5 plate reader (Molecular Devices, Inc) using the following settings: excitation - 485 nm, emission - 525 nm, cutoff filter - 515 nm, medium sensitivity. The measurements were taken at room temperature every 69 sec for 1h.
5) The progress curves were analyzed using linear regression within SoftMax Pro software to obtain the initial slope of the curve and R-squared parameter. The curves with R-squared below 0.98 were visually inspected for non-linearity and the linearization range was adjusted to reflect the initial slope.
6) The initial slope of the progress curves was plotted vs. compound concentration and the resulting plots were analyzed using a 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 MKP-3 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 equationString
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 Float
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 Float
31IC50_Qualifier_8This qualifier is to be used with the next TID, IC50_8. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
32IC50_8IC50 value determined using sigmoidal dose response equationFloatμM
33Std.Err(IC50)_8Standard Error of IC50 valueFloatμM
34nH_8Hill coefficient determined using Float
35IC50_Qualifier_9This qualifier is to be used with the next TID, IC50_9. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
36IC50_9IC50 value determined using sigmoidal dose response equationFloatμM
37Std.Err(IC50)_9Standard Error of IC50 valueFloatμM
38nH_9Hill coefficient determined using Float
39IC50_Qualifier_10This qualifier is to be used with the next TID, IC50_10. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
40IC50_10IC50 value determined using sigmoidal dose response equationFloatμM
41Std.Err(IC50)_10Standard Error of IC50 valueFloatμM
42nH_10Hill coefficient determined using Float
43IC50_Qualifier_11This qualifier is to be used with the next TID, IC50_11. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
44IC50_11IC50 value determined using sigmoidal dose response equationFloatμM
45Std.Err(IC50)_11Standard Error of IC50 valueFloatμM
46nH_11Hill coefficient determined using Float
47IC50_Qualifier_12This qualifier is to be used with the next TID, IC50_12. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
48IC50_12IC50 value determined using sigmoidal dose response equationFloatμM
49Std.Err(IC50)_12Standard Error of IC50 valueFloatμM
50nH_12Hill coefficient determined using Float
51IC50_Qualifier_13This qualifier is to be used with the next TID, IC50_13. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
52IC50_13IC50 value determined using sigmoidal dose response equationFloatμM
53Std.Err(IC50)_13Standard Error of IC50 valueFloatμM
54nH_13Hill coefficient determined using Float
55IC50_Qualifier_14This qualifier is to be used with the next TID, IC50_14. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
56IC50_14IC50 value determined using sigmoidal dose response equationFloatμM
57Std.Err(IC50)_14Standard Error of IC50 valueFloatμM
58nH_14Hill coefficient determined using Float
59IC50_Qualifier_15This qualifier is to be used with the next TID, IC50_15. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value.String
60IC50_15IC50 value determined using sigmoidal dose response equationFloatμM
61Std.Err(IC50)_15Standard Error of IC50 valueFloatμM
62nH_15Hill coefficient determined using Float

* Activity Concentration.
Additional Information
Grant Number: XO1 MH076390-01

Data Table (Concise)
Classification
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