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In vitro MKP-1 Phosphatase Dose Response Active/Probe Assessment Assay - Effects of DTT

PubChem AID
558
Source
University of Pittsburgh Molecular Library Screening Center
External ID
MH-76391 MLSCN MKP-1 Dose Response Active/Probe Assessment -DTT
BioAssay Type
Confirmatory
Tested Substances
Version
Status
Live
Dates
  • Deposit:
    2006-12-18
  • Modify:
    2006-12-19
Description
Please note that the bioassay record (AID 558) is presented as provided to PubChem by the source(depositor). When possible, links to additional information have been provided by PubChem.

1 Description

The MKP-1 dose response Active/Probe assessment-DTT assay has been developed to evaluate the effects of increased DTT concentration on the MKP-1 inhibition of actives identified in the MH-76391 In vitro MKP-1 HTS assay AID 374, and subsequently confirmed in the HTS dose

response confirmation assay AID 551. Protein tyrosine phosphatases have an active site cysteine that is very susceptible to inactivation by oxidation. In addition, a number of compounds such as quinone-like compounds are capable of generating reactive oxygen species via redox cycling in the presence of DTT. Increasing the concentration of DTT from 1 mM to 25 mM does not affect the activity of MKP-1 in the assay but can reverse the inhibition of some inhibitors or significantly increase their IC50 values. The MKP-1 Phosphatase Dose Response Active/Probe Assessment Assay-Effects of DTT has been Developed and Run at the University of Pittsburgh Molecular Screening Center (PMLSC) part of the Molecular Library Screening Center Network (MLSCN). XO1 submission MH-76391 In vitro HTS assay for MKP-1, Assay Provider Dr. John S. Lazo, Department of Pharmacology at the University of Pittsburgh.

The 107 protein tyrosine phosphatases (PTPs) found in the human genome are defined by the active site sequence C(X)5R(S/T), with X being any amino acid, and they are critical regulators of mammalian cell proliferation, differentiation, and apoptosis. PTPs can be divided into at least four subfamilies based on their tertiary structure: Classical phosphotyrosine specific PTPs, dual specificity phosphatases (DSPase), Cdc25 phosphatases and low molecular weight PTPs. One subgroup of the DSPase family are the MAPK phosphatases (MKP), which dephosphorylate MAPKs on threonine and tyrosine residues. Mitogen-activated protein kinase phosphatase-1 (MKP-1, DUSP1 or CL100) dephosphorylates and inactivates MAPK substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. MKP-1 is highly expressed in prostate, breast, gastric, and renal cancer. In patients with ovarian cancer, MKP-1 expression is correlated with decreased progression-free survival. Liao et al. reported that reduction of MKP-1 in human pancreatic cells ablated soft agar colony formation and tumorigenicity. Others have found MKP-1 protects cells from apoptosis induced by cisplatin, UV irradiation, and proteasome inhibitors. The association of MKP-1 with neoplasia makes it an attractive therapeutic target, however unlike other phosphatases such as PTP1B, there is a lack of readily available selective small molecule inhibitors for MKP family members.

One subgroup of the DSPase family are the MAPK phosphatases (MKP), which dephosphorylate MAPKs on threonine and tyrosine residues. Mitogen-activated protein kinase phosphatase-1 (MKP-1, DUSP1 or CL100) dephosphorylates and inactivates MAPK substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. MKP-1 is highly expressed in prostate, breast, gastric, and renal cancer. In patients with ovarian cancer, MKP-1 expression is correlated with decreased progression-free survival. Liao et al. reported that reduction of MKP-1 in human pancreatic cells ablated soft agar colony formation and tumorigenicity. Others have found MKP-1 protects cells from apoptosis induced by cisplatin, UV irradiation, and proteasome inhibitors. The association of MKP-1 with neoplasia makes it an attractive therapeutic target, however unlike other phosphatases such as PTP1B, there is a lack of readily available selective small molecule inhibitors for MKP family members.

The MKP-1 assay is a 384-well format homogeneous fluorescence intensity assay measuring the hydrolysis of the generic substrate 3-O-methylfluorescein phosphate (OMFP). His-tagged MKP-1 was expressed in BL21(DE3) competent bacterial cells transformed with the pET21a-MKP-1 expression vector and induced with IPTG. Biologically active MKP-1 was partially purified from IPTG-induced bacterial cell lysates by Ni-column chromatography eluted with imidazole, frozen, aliquoted and stored at 80 degrees C. The MKP-1 assay conforms to the PMLSC assay development and implementation guidelines.

2 Protocol

Materials:

1.10X Assay Buffer: 300 mM Tris, 750 mM NaCl, 10 mM EDTA, pH 7.0.

Trizma 47.28 g + NaCl 43.83 g +EDTA 3.722 g + dH2O 800mL, pH to 7.0 with NaOH, add dH2O to 1000mL.

2.DTT: 200 mM Stock in -20 ◦C. The final concentration of DTT has been increased from 1 mM to 25 mM to test the effects of DTT on MKP-1 inhibition.

3.BSA: 6.6% BSA Stock in -20 ◦C.

4.OMFP Substrate: 5.0 uL/well ~ 40 uM final (1% DMSO).

Add 1.05 mg OMFP per 0.5 mL of DMSO and sonicate in water bath ~ 5 min to make a 4.0 mM stock of OMFP (100% DMSO). Immediately prior to the assay dilute the 4.0 mM OMFP to 120 uM in dH2O and transfer 5 uL to each well of a 384-well of the assay plate ~ 40 uM OMFP final (1% DMSO).

5.MKP-1 Enzyme: 5.0 uL/well ~ 250 ng/well.

Partially purified MKP-1 enzyme at 685 ug/mL in 0.5 mL aliquots stored at -80 ◦C.

6.Compounds: 5.0 uL/well ~ 10 to 100 uM final (1% DMSO).

Dilute compounds in dH2O to the appropriate concentration (30 to 300 uM, 3% DMSO) and transfer 5 uL per well.

7.Maximum Control: 5.0 uL/well of 3% DMSO in dH2O

8.Minimum Control: 5.0 uL/well of 300 mM NaOH in 3% DMSO in dH2O.

9.50 % Control: 5.0 uL/well of 60 mM NaOH in 3% DMSO in dH2O.

10.Assay stop solution: 5.0 uL/well of a 500 mM NaOH stock (125 mM final).

Max controls: 250 ng MKP-1 + 40 uM OMFP + 2% DMSO

Min controls: 250 ng MKP-1 + 40 uM OMFP + 2% DMSO + 100 mM NaOH

50 % controls: 250 ng MKP-1 + 40 uM OMFP + 2% DMSO + 20 mM NaOH

MKP-1 Assay Protocol:

1.Transfer 5 uL of diluted compounds and plate controls to wells of 384-well black low-volume 384-well assay plate, Greiner # 784076.

2.Transfer 5 uL/well of 3x MKP-1 enzyme solution (75 mM DTT)to assay plate.

3.Transfer 5 uL/well of 120 uM OMFP solution to assay plate.

4.Centrifuge plate at 500 RPM's 20 secs.

5.Incubate assay plate at ambient temperature with shaking for 60 min.

6.Stop assay by transfer of 5 uL/well 500 mM NaOH to assay plate.

7.Read fluorescence intensity at Ex 485 nM Em 525 nM on the M5 plate reader.

3 Comment

List of compounds to be tested.

Activies (≥ 50% inhibition) identified in the in vitro MKP-1 HTS and subsequently confirmed in the HTS dose response confirmation assay.

PMLSC IC50 Confirmation Scoring System:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when IC50 is > 50 uM

2 - Substance is considered active when IC50 is < 50 uM

3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

40 - Compounds that were active in the HTS but failed to confirm in the 10-pt dose response assay with an IC50 > 50 uM

50 - Compounds that were active in the HTS and confirmed in the 10-pt dose response assay with an IC50 in the 20 to 50 uM range

60 - Compounds that were active in the HTS and confirmed in the 10-pt dose response assay with an IC50 in the 10 to 20 uM range

70 - Compounds that were active in the HTS and confirmed in the 10-pt dose response assay with an IC50 in the 1 to 10 uM range

80 - Compounds that were active in the HTS and confirmed in the 10-pt dose response assay with an IC50 < 1 uM

Data Analysis:

Compounds will be tested in singlicate 10-point dose response assays, typically at a maximum concentration of 50uM final (1% DMSO). Compounds will be diluted to 150 uM in dH2O (3% DMSO final) and serially diluted in dH2O + 3% DMSO to provide the desired concentration range. 5uL of the compound dilutions will be transferred to a total of 15 uL assay volume (3-fold dilution)

An ActivityBase template has been written that will automatically calculate % inhibition together with plate control Signal to Noise and Z-factors for secondary dose response data. These data will be used to perform quality control (QC) and either pass or fail plates. For data from plates that pass QC, IC50#s will be calculated using the four parameter logistic model, also called the Sigmoidal Dose-Response Model. One form of the equation is:

y = (A+((B-A)/(1+((x/C)^D))))

where y is the percent inhibition and x is the corresponding concentration. The fitted C parameter is the IC50, and is defined as the concentration giving a response half way between the fitted top and bottom of the curve. The fitted D parameter is called the #Slope#. The A and B parameters are locked. A is assigned to be 0 and B is 100. Also reported is an R2 value; the square of the linear correlation coefficient for a given fit cell. It will return a value between 0 and 1.00 as the correlation coefficient only returns values between -1.00 and 1.00.

4 Result Definitions

5 Data Table

6 Target

9 Identity

9.1 BioAssay Name

In vitro MKP-1 Phosphatase Dose Response Active/Probe Assessment Assay - Effects of DTT

9.2 Source

University of Pittsburgh Molecular Library Screening Center

9.3 External ID

MH-76391 MLSCN MKP-1 Dose Response Active/Probe Assessment -DTT

9.4 Project Category

NIH Molecular Libraries Screening Center Network

9.5 BioAssay Type

Confirmatory

9.6 Deposit Date

2006-12-18

9.7 Modify Date

Version 1.1
Version 2.1
2006-12-19 (currently shown)

9.8 Status

Live

10 Same-Project BioAssays

11 BioAssay Annotations

Assay Format
cell-based format
Assay Type
direct enzyme activity assay
Detection Method
fluorescence intensity

12 Information Sources

CONTENTS