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BioAssay: AID 652006

Dose response confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, in a fluorescence-based, Lymphoid Phosphatase (PTPN22, LYP-1) selectivity Assay

Assay Provider: Nunzio Bottini, M.D., Ph.D., La Jolla Institute for Allergy & Immunology, La Jolla, CA ..more
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 Tested Compounds
 Tested Compounds
All(71)
 
 
Active(9)
 
 
Inactive(62)
 
 
 Tested Substances
 Tested Substances
All(71)
 
 
Active(9)
 
 
Inactive(62)
 
 
AID: 652006
Data Source: Burnham Center for Chemical Genomics (SBCCG-A956-LMPTP-Inh-DMSO-DR-LYP-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 9
Related Experiments
AIDNameTypeComment
651560uHTS identification of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assayScreeningdepositor-specified cross reference
651562Summary assay for small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTPSummarydepositor-specified cross reference
651700Dose response confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assayConfirmatorysame project related to Summary assay
652005Dose response confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, in an orthogonal absorbance-based assayConfirmatorysame project related to Summary assay
686961Dose response confirmation of uHTS small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, in a fluorescence-based, VHR-1 (dual specificity phosphatase 3) selectivity assayConfirmatorysame project related to Summary assay
686962SAR confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assayConfirmatorysame project related to Summary assay
686963SAR confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, in an orthogonal absorbance-based assayConfirmatorysame 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 Production Centers Network (MLPCN)
Grant Number: 1 R03 DA033986-01
Assay Provider: Nunzio Bottini, M.D., Ph.D., La Jolla Institute for Allergy & Immunology, La Jolla, CA

Obesity is frequently complicated by a constellation of metabolic and cardiovascular anomalies, called the metabolic syndrome, which significantly increases morbidity and mortality of affected individuals. Insulin resistance is an important component of the metabolic syndrome. Protein tyrosine phosphatases (PTPs) that regulate insulin signaling are, in principle, excellent therapeutic targets for insulin resistance syndromes. Indeed, PTP1B, a critical negative regulator of insulin signaling in liver and skeletal muscle, is currently an important drug target in obesity and type 2 diabetes. This grant proposal focuses on another PTP, the low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene. LMPTP is highly expressed in adipocytes. There is strong in vitro and in vivo evidence that LMPTP is a negative regulator of insulin signaling and a promising drug target in obesity. Genetic association studies in humans support a negative role for LMPTP in insulin resistance and the metabolic complications of obesity. In vivo, partial knock-down of LMPTP expression by specific antisense oligonucleotides (ASOs) led to improved glycemic and lipid profiles and decreased insulin resistance in diet-induced obese C57BL/6 mice. Interestingly, anti-LMPTP ASOs did not induce any metabolic phenotype in lean mice. Our current working model is that LMPTP plays a critical negative role in adipocyte insulin signaling, while it is less important in liver and muscle, where it can be at least partially compensated for by PTP1B and/or other prominent PTPs. We hypothesize that a specific small-molecule inhibitor of LMPTP will significantly reduce obesity associated insulin resistance and decrease the severity of the metabolic syndrome in obesity.

The goal of this assay is to assess the selectivity of compounds identified in "uHTS identification of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assay" (AID 651560) in dose response against Lymphoid Phosphatase (LYP-1), also known as protein tyrosine phosphatase, non-receptor type 22 (PTPN22), a related enzyme. This is accomplished via an enzymatic reaction utilizing a fluorogenic phosphatase substrate.
Protocol
Protocol
A. Brief Description of the Assay:
This assay attempts to determine the selectivity of compounds identified in the primary screen as potential inhibitors of the LMPTP-A (Low Molecular Weight Protein Tyrosine Phosphatase-A) enzyme by measuring the activity against the LYP-1 enzyme. The assay is run in 1536-well format and is measured via fluorescence.

B. Materials:
Item, source, catalog no.
His-LYP Catalaytic Domain Enzyme Stock Solution, 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 High base opaque bottom plate, Nexus Biosystems, 00019120

C. Final Assay Conditions:
Reagent, Final Concentration
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 1.0 mM
His-LYP, 2.5 nM
OMFP, 770 uM
BSA, 0.1%
6uL reaction volume
50 minutes incubation at room temp
D. Assay Procedures:
1. Prepare Reagents as described in section F. Recipe.
2. Using LabCyte Echo, transfer varying volumes of compounds in 10 mM DMSO to achieve appropriate dose concentrations and range (Col. 5-48). Back fill compound wells and control wells to equilibrate DMSO concentrations across plate.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Using the Multidrop Combi, add 2.5 uL/well of control buffer to columns 1 and 2.
5. Using the Multidrop Combi, add 2.5 uL/well of enzyme solution to col. 3-48.
6. Using the Multidrop Combi, add 2.5 uL/well of substrate solution to col. 1-48.
7. Spin plates at 1000 rpm for 1 minute in centrifuge.
8. Incubate plates in the dark at room temperature for 50 minutes.
9. Read plates on PerkinElmer Viewlux using a FI protocol.

E. Plate Map:
Positive (Low) control in column 1-4, DMSO, substrate only.
Negative (High) control in columns 45-48, DMSO, enzyme and substrate.
Test wells in columns 5-44, test compound, enzyme and substrate.

F. Recipe:
1X Assay Buffer
50 mM Bis-Tris pH 6.0
1 mM DTT
0.005% Tween-20
0.1% BSA

Control Buffer
1X Assay Buffer

Enzyme Solution
5.0 nM LYP-1 Enzyme in 1X Assay Buffer (final enzyme concentration is 2.5 nM).

Substrate Solution
1540 uM OMFP Substrate in 1X Assay Buffer (final substrate concentration is 770 nM).

G. Note:
All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
Storage conditions after reagents are made up:
Reagent, Temp
Bis Tris pH 6.0, 4 degrees
His-LYP protein stock solution, -80 degrees
OMFP, -80 degrees (light sensitive)
Tween-20, -80 degrees
Comment
Compounds that demonstrated an IC50 <= 20 uM are defined as actives in the assay.

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 and is not applicable in this assay

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 and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. 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 target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
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 high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
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 the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 80 uM_first_point (80μM**)% Activity at the test concentrationFloat%
9% Activity at 40 uM_first_point (40μM**)% Activity at the test concentrationFloat%
10% Activity at 20 uM_first_point (20μM**)% Activity at the test concentrationFloat%
11% Activity at 10 uM_first_point (10μM**)% Activity at the test concentrationFloat%
12% Activity at 5 uM_first_point (5μM**)% Activity at the test concentrationFloat%
13% Activity at 2.5 uM_first_point (2.5μM**)% Activity at the test concentrationFloat%
14% Activity at 1.25 uM_first_point (1.25μM**)% Activity at the test concentrationFloat%
15% Activity at 0.625 uM_first_point (0.625μM**)% Activity at the test concentrationFloat%
16% Activity at 0.3125 uM_first_point (0.3125μM**)% Activity at the test concentrationFloat%
17% Activity at 0.15625 uM_first_point (0.15625μM**)% Activity at the test concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 80 uM_second_point (80μM**)% Activity at the test concentrationFloat%
20% Activity at 40 uM_second_point (40μM**)% Activity at the test concentrationFloat%
21% Activity at 20 uM_second_point (20μM**)% Activity at the test concentrationFloat%
22% Activity at 10 uM_second_point (10μM**)% Activity at the test concentrationFloat%
23% Activity at 5 uM_second_point (5μM**)% Activity at the test concentrationFloat%
24% Activity at 2.5 uM_second_point (2.5μM**)% Activity at the test concentrationFloat%
25% Activity at 1.25 uM_second_point (1.25μM**)% Activity at the test concentrationFloat%
26% Activity at 0.625 uM_second_point (0.625μM**)% Activity at the test concentrationFloat%
27% Activity at 0.3125 uM_second_point (0.3125μM**)% Activity at the test concentrationFloat%
28% Activity at 0.15625 uM_second_point (0.15625μM**)% Activity at the test concentrationFloat%
29IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2IC50 value determined using a sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2Standard Error of the IC50 valueFloatμM
32nH_2Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 80 uM_third_point (80μM**)% Activity at the test concentrationFloat%
35% Activity at 40 uM_third_point (40μM**)% Activity at the test concentrationFloat%
36% Activity at 20 uM_third_point (20μM**)% Activity at the test concentrationFloat%
37% Activity at 10 uM_third_point (10μM**)% Activity at the test concentrationFloat%
38% Activity at 5 uM_third_point (5μM**)% Activity at the test concentrationFloat%
39% Activity at 2.5 uM_third_point (2.5μM**)% Activity at the test concentrationFloat%
40% Activity at 1.25 uM_third_point (1.25μM**)% Activity at the test concentrationFloat%
41% Activity at 0.625 uM_third_point (0.625μM**)% Activity at the test concentrationFloat%
42% Activity at 0.3125 uM_third_point (0.3125μM**)% Activity at the test concentrationFloat%
43% Activity at 0.15625 uM_third_point (0.15625μM**)% Activity at the test concentrationFloat%
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 80 uM_fourth_point (80μM**)% Activity at the test concentrationFloat%
46% Activity at 40 uM_fourth_point (40μM**)% Activity at the test concentrationFloat%
47% Activity at 20 uM_fourth_point (20μM**)% Activity at the test concentrationFloat%
48% Activity at 10 uM_fourth_point (10μM**)% Activity at the test concentrationFloat%
49% Activity at 5 uM_fourth_point (5μM**)% Activity at the test concentrationFloat%
50% Activity at 2.5 uM_fourth_point (2.5μM**)% Activity at the test concentrationFloat%
51% Activity at 1.25 uM_fourth_point (1.25μM**)% Activity at the test concentrationFloat%
52% Activity at 0.625 uM_fourth_point (0.625μM**)% Activity at the test concentrationFloat%
53% Activity at 0.3125 uM_fourth_point (0.3125μM**)% Activity at the test concentrationFloat%
54% Activity at 0.15625 uM_fourth_point (0.15625μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA033986-01

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