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BioAssay: AID 588621

uHTS identification of small molecule inhibitors of Striatal-Enriched Phosphatase via a fluorescence intensity assay

Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation is crucial for the development of many serious conditions, including cancer, diabetes, and autoimmune disorders. This is the first time that tyrosine phosphatase inhibitors are being proposed to improve cognitive function in Alzheimer's disease (AD). STriatal-Enriched Phosphatase (STEP) is a more ..
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 Tested Compounds
 Tested Compounds
All(359231)
 
 
Active(887)
 
 
Inactive(358344)
 
 
 Tested Substances
 Tested Substances
All(359521)
 
 
Active(889)
 
 
Inactive(358632)
 
 
AID: 588621
Data Source: Burnham Center for Chemical Genomics (SBCCG-A754-STEP-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-24
Modify Date: 2011-10-25

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 887
Depositor Specified Assays
AIDNameTypeComment
588619Summary assay for small molecule inhibitors of Striatal-Enriched Phosphatasesummary
602367Dose Response selectivity of inhibitors of Striatal-Enriched Phosphatase (STEP) in a SHP2 (PTPN11) Inhibition Assayconfirmatory
602372Dose response confirmation of uHTS small molecule inhibitors of Striatal-Enriched Phosphatase via a fluorescence intensity assayconfirmatory
602374Dose Response selectivity of inhibitors of STriatal-Enriched Phosphatase (STEP) in the dual-specificity protein-tyrosine phosphatase VHR Inhibition Assayconfirmatory
623866Single concentration confirmation molecule inhibitors of Striatal-Enriched Phosphatase via a fluorescence intensity assayscreening
624207Dose response orthogonal assay of uHTS small molecule inhibitors of Striatal-Enriched Phosphatase via a colorimetric intensity assay.confirmatory
624241Dose Response selectivity of inhibitors of STriatal-Enriched Phosphatase (STEP) in the Lymphoid Phosphatase (PTPN22) Inhibition Assayconfirmatory
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 Probe Production Centers Network (MLPCN)
Grant Number: 1R03MH095532-01
Assay Provider: Dr. Lutz Tautz, Sanford-Burnham Medical Research Institute, San Diego CA

Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation is crucial for the development of many serious conditions, including cancer, diabetes, and autoimmune disorders. This is the first time that tyrosine phosphatase inhibitors are being proposed to improve cognitive function in Alzheimer's disease (AD). STriatal-Enriched Phosphatase (STEP) is a brain-specific protein tyrosine phosphatase that is highly expressed in regions where consolidation of memory occurs and regulates the internalization of NMDARs. Our recent work demonstrates that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Moreover, using genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model, we showed that a decrease in STEP levels attenuates the cognitive and cellular deficits observed in six-month old 3xTg-AD mice. The hypothesis that STEP inhibitors may prove therapeutic for the treatment of AD is a shift in the current paradigm of reducing A-beta levels to inhibiting a downstream target of A-beta. Recent data suggest that STEP inhibitors may prove therapeutic for this devastating disorder.

The goal of this high-throughput assay is to identify hit compounds for the STriatal-Enriched Phosphatase (STEP). This is accomplished via an enzymatic reaction utilizing a fluorogenic phosphatase substrate.
Protocol
STEP Assay HTS Protocol

A. Brief Description of the Assay:
This assay idenitfies inhibitors of STEP (STriatal-Enriched Phosphatase) enzyme. It is measured via fluorescence in 1536-well plate format.

B. Materials:
Item, Source, Cat #
STEP Enzyme Stock Solution 6.2mg/mL (178uM), Dr. Lutz Tautz, N/A
Bis-Tris, Fisher Sci, BP301-100
Tween 20, Sigma, P1379
DTT, Sigma, D9779
OMFP, Sigma, M2629-100MG
Mol. Grade Water, Mediatech, Inc., 46-000-CM
1536 well black solid flat bottom Non-Binding plate, Corning, 3724

C. Final Assay Conditions:
Reagent, Final Concentration
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 2.5 mM
STEP, 0.5 nM
OMFP, 25 uM
Final reaction volume, 4 uL/well in 1536 well plate
Test compound concentration, 20 uM
Final DMSO concentration, 1.0%
D. Procedures:
Step#, Description
1. Prepare Reagents as described in sections F. Recipe.
2. Using LabCyte Echo, transfer 40 nL from a plate containing 2 mM test compounds into assay plate Col. 5 - 44 (final concentration of test compounds is 20 uM, 1.0% DMSO). 40nL of DMSO should be transferred to col. 1-4 for control wells.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Set up Kalypsys dispenser as described in section G. Instrument settings.
5. Using the Kalypsys dispenser, add 2 uL/well of control buffer (no enzyme control) to columns 1 and 2 for the positive control wells.
6. Using the Kalypsys dispenser, add 2 uL/well of enzyme solution to col. 3-48 for the negative control and test compound wells.
7. Using the Kalypsys dispenser, add 2 uL/well of substrate solution to columns. 1-48 (all wells).
8. Spin plates at 1000 rpm for 1 minute in centrifuge.
9. Incubate plates in the dark at room temperature for 20 minutes.
10. Detect signals on Perkin Elmer Viewlux with settings as described in section G. Instrument settings.

E. Plate Map:
Positive (Low) control in columns 1 - 2, DMSO, substrate only
Negative (High) control in columns 3 and 4, DMSO, enzyme and substrate
Test compound in columns 5 - 48, Test compounds, enzyme and substrate

F. Recipe:
Enzyme solution (STEP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 5 mM
STEP, 0.5 nM

Substrate solution (OMFP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
OMFP, 50uM

G. Instrument settings:
Kalypsys dispenser
Step#, Description
1. Before assay starts, rinse tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
2. Air rinse tubing.
3. Rinse and prime tubing with 1 mL of actual reagents per dispensing tip.
4. When the assay is done, clean tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
5. Air rinse tubing.
6. Rinse tubing thoroughly with 5 mL of 25% EtOH per dispensing tip.

Perkin Elmer Viewlux
Light Energy: 10000
Measurement: Time 1 sec.
Excitation Filter: 480/20 (FITC)
Emission Filter: 540/25 (FITC)
Mirror: FITC dichroic
Sensitivity: 4.13 e - /ADU

H. Note:
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Make up buffer minus Tween-20 in large scale and add fresh Tween-20 weekly.
3. Make up enzyme buffer minus DTT in large scale and add fresh DTT just before the assay starts.
4. Storage conditions after reagents are made up:
Reagent, Temp.
Buffer minus DTT, 4 degree
STEP, -80 degrees
Na3VO4, -80 degrees
DTT, -80 degrees
OMFP, -80 degrees (light sensitive)

The experimental values were normalized by difference between values from neutral and stimulator control wells in each plates. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing and edge effect due to overnight incubation. The algorism "Assay Pattern (Multiplicative)" was applied in Genedata Screener(R) software to correct screen data. Further information about data correction is available at http://www.genedata.com/products/screener.html.
Compounds that demonstrated % activity of >= 40 % at 20 uM
Comment
Compounds that demonstrated a normalized or corrected inhibition of >= 40% at 20uM concentration are defined as actives in this assay.

The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.

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.

Activity Scoring
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 assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 20 uM_Norm (20μM**)Normalized % inhibition in primary screeningFloat%
2%Activity at 20 uM_Corr (20μM**)Genedata pattern corrected % inhibition in primary screeningFloat%
3Value (20μM**) Value of the sample FloatRFU
4Mean Low Mean fluorescent signal of positive controls in the corresponding plate FloatRFU
5Std Deviation Low Standard deviation (n=64) of positive controls in the corresponding plate FloatRFU
6Mean High Mean fluorescent signal of negative controls in the corresponding plate FloatRFU
7Std Deviation High Standard deviation (n=64) of negative controls in the corresponding plate FloatRFU

** Test Concentration.
Additional Information
Grant Number: 1R03MH095532-01

Data Table (Concise)
Classification
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