Dose response confirmation of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, in an orthogonal absorbance-based assay
Assay Provider: Nunzio Bottini, M.D., Ph.D., La Jolla Institute for Allergy & Immunology, La Jolla, CA ..more
BioActive Compounds: 73
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 test activity in dose response in an orthogonal assay for compounds identified in "uHTS identification of small molecule inhibitors of Low Molecular Weight Protein Tyrosine Phosphatase, LMPTP, via a fluorescence intensity assay" (AID 651560). This is accomplished via an enzymatic reaction utilizing a colorimetric substrate.
A. Brief Description of the Assay:
This assay attempts to confirm in an orthogonal assay, via dose dependent response, compounds identified in the primary screen as potential inhibitors of the LMPTP-A (Low Molecular Weight Protein Tyrosine Phosphatase-A) enzyme. The assay is run in 1536-well format and is measured via absorbance.
Item, source, catalog no.
LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 uM), SBMRI Protein Facility, N/A
pNPP - 20 mg tablets, Sigma, N2765
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
NaOH, Fisher Scientific, SS267
1536-well clear plates, Greiner, 782101
C. Final Assay Conditions:
1.25 nM LMPTP-A Enzyme
400 uM OMFP
50 mM Bis-Tris pH 6.0
1 mM DTT
0.01% Triton-X 100
2.8% DMSO (2% from substrate and 0.8% from compounds)
6 uL 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. After the incubation, add 5 uL/well of the Stop Reagent to col. 1-48 to stop the reaction.
10. Spin plates at 1000 rpm for 1 minute in centrifuge.
10. Read plates on a Pherastar or other appropriate platereader for absorbance at 405 nm.
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.
1X Assay Buffer
50 mM Bis-Tris pH 6.0
1 mM DTT
0.01% Triton-X 100
1X Assay Buffer
2.5 nM LmPTP-A Enzyme in 1X Assay Buffer (final enzyme concentration is 1.25 nM).
14 nM pNPP Substrate in 1X Assay Buffer (final substrate concentration is 7 nM).
200 mM NaOH
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
pNPP, -80 degrees (light sensitive)
DTT, -80 degrees
Triton-X 100, Room Temp
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 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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)