uHTS identification of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assay
Assay Provider: Nunzio Bottini, M.D., Ph.D., La Jolla Institute for Allergy & Immunology, La Jolla, CA ..more
BioActive Compounds: 817
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.
Our objective of this endeavor is to screen the NIH chemical library for novel inhibitors of the LMPTP. The focus of the assay will be the identification of non-competitive allosteric inhibitors. This will be accomplished via the use of a fluorogenic substrate of LMPTP.
A. Brief Description of the Assay:
This assay attempts to identify inhibitors of the LMPTP-A (Low Molecular Weight Protein Tyrosine Phosphatase-A) enzyme. It is run in 1536-well format and is measured via fluorescence intensity.
Item, source, catalog no.
LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8uM), SBMRI Protein Facility, N/A
OMFP, Sigma, M2629-100MG
Bis-Tris pH 6.0, Fisher Sci, BP301-100
Triton-X 100, Sigma, T9284
DTT, Sigma, D9779
Mol. Grade Water, Mediatech, Inc., 46-000-CM
1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120
C. Final Assay Conditions:
0.625nM LMPTP-A Enzyme
50mM Bis-Tris pH 6.0
0.01% Triton-X 100
20uM test compound
3% DMSO (2% from substrate and 1% from compounds)
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 60 nL from 2mM test compound source plate into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM). 60nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Using the Beckman Coulter Bioraptr, add 3 uL/well of control buffer to columns 1 and 2.
5. Using the Bioraptr, add 3 uL/well of enzyme solution to col. 3-48.
6. Using the Bioraptr, add 3 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, DMSO, substrate only.
Negative (High) control in columns 3-4, DMSO, enzyme and substrate.
Test wells in columns 5-48, 20uM test compound, enzyme and substrate.
1X Assay Buffer
50mM Bis-Tris pH 6.0
0.01% Triton-X 100
1X Assay Buffer
1.25nM LmPTP-A Enzyme in 1X Assay Buffer (final enzyme concentration is 0.625nM).
800uM OMFP Substrate in 1X Assay Buffer (final substrate concentration is 400uM).
All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
Storage conditions after reagents are made up:
Bis Tris pH 6.0, 4 degrees
LMPTP-A, -80 degrees
OMFP, -80 degrees (light sensitive)
DTT, -80 degrees
Triton-X 100, Room Temp
Compounds that demonstrated a normalized or corrected % activity of <=50% compared to the controls are defined as inhibitors of the reaction.
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 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
** Test Concentration.
Data Table (Concise)