|Dose Response confirmation of Image-Based HTS for Selective Agonists for NTR1 - BioAssay Summary
Addiction leading to abuse should be treatable by pharmacological approaches, and programs that identify new drugs to treat methamphetamine abuse address an immediate goal of the National Institute on Drug Abuse (NIDA) that new approaches are needed for treating METH addiction. Currently, small molecule drug-like compounds are not available for treating METH abuse. Neurotensin receptor 1 (NTR1) more ..
BioActive Compounds: 138
Depositor Specified Assays
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 Proposal Number: 1 R03 MH089653-01
Assay Provider: Dr. Lawrence Barak, Duke University Medical Center
Addiction leading to abuse should be treatable by pharmacological approaches, and programs that identify new drugs to treat methamphetamine abuse address an immediate goal of the National Institute on Drug Abuse (NIDA) that new approaches are needed for treating METH addiction. Currently, small molecule drug-like compounds are not available for treating METH abuse. Neurotensin receptor 1 (NTR1) peptide agonists produce behaviors that are exactly opposite to the psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes, and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance . These data form the basis of the idea that neurotensin receptors are valid targets for antagonizing drug seeking behaviors and preventing relapses. We propose to identify novel small molecule neurotensin receptor agonists by screening libraries of compounds using a primary assay based upon the ability of a b-arrestin fluorescent reporter to directly recognize the activated state of the NTR1.
The goal of this assay is to confirm hits from "Image-Based HTS for Selective Agonists for NTR1", (AID 493036).
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A. Brief Description of the Assay:
This assay is to perform a 10 pt. dose-response on confirmed lead agonists that activate the Neurotensin receptor-1 (NTR1) pathway by detecting spots from beta-arrestin GFP in NTR1-U2OS Osteosarcoma Cells.
Description Source Cat
NTR1-U2OS osteosarcoma cells Dr. Lawrence Barak, Duke University N/A
MEM medium Cellgro/Mediatech 15-010-CM
Fetal Bovine Serum Hyclone SH30396.03
Penicillin Streptomycin solution Omega Scientific PS-20
L-Glutamine Mediatech 25-005-CL
G418 (100 mg/mL) Invivogen ant-gn-1
Zeocin (100 mg/mL) Invitrogen R250-01
T225 Tissue Culture flasks Corning 431082
DPBS Cellgro 21-031CV
Trypsin-EDTA 0.05% Invitrogen 25300
1536 well black clear bottom plate Aurora Biotechnology 29326
NTR1 peptide Sigma N6383
Paraformaldehyde Acros Organics 30528954
Hoechst 33342 (10 mg/mL) Invitrogen H3570
Aluminum Plate Seals E&K Scientific T592100
C. Plate Map:
Positive (High) control (P) in columns 3, 4, 45 and 46, DMSO and 100 nM NTR1
Negative (Low) control (N) in columns 1, 2, 47 and 48, DMSO but No NTR1
Test compound in columns 5 - 48, Test compound but No NTR1
1 Prepare cell suspension as described in section F. Cell Culture.
2 Set up and prime Combi liquid dispenser
3 Dispense 8 uL/well of cells at 7X10^10 cells/mL using Combi into a black 1536 well plate with clear bottom.
4 Spin down plates on Eppendorf 5810 centrifuge at 1000 rpm for 1 minute.
5 Put Kalypsys metal lids on plates, and incubate plates at 37 oC with 5% CO2 overnight.
1 Set up Combi and nL liquid dispensers, Kalypsys plate washer and Opera.
2 Transfer 40, 20, 10, 5, and 2.5 nL/well of 10 mM and 315uM test compounds into assay plates using the Labcyte ECHO 555. The final concentration of compounds starts at 40uM diluted half-fold to 78nM. Compounds are screened in triplicates.
3 Add 4 uL/well of 300 nM NTR1 peptide in DPBS to columns 3, 4, 45 and 46 of the assay plates and then add 4 uL/well of DPBS (without NTR1 peptide) to columns 1, 2, 5-44, 47, and 48 using Combi. The final concentration of NTR1 peptide is 100nM.
4 Spin down plates on Eppendorf 5810 centrifuge at 1000 rpm for 1 minute.
5 Incubate plates at 37 oC with 5% CO2 for 1 hour.
6 Fix cells by adding 4 uL/well of 6% Paraformaldehyde into all wells using Combi. The final concentration of Paraformaldehyde is 1.7%.
7 Spin down plates on Eppendorf 5810 centrifuge at 1000 rpm for 1 minute, and incubate for one hour at room temperature.
8 Wash plates with 1X DPBS using the Kalypsys plate washer. The wash program consists of aspirating steps that leave ~2.5 uL/well and dispensing 11 uL/well DPBS at two cycles, and eventually leaving ~2.5 uL/well.
9 Add 5 uL/well of 5 ug/mL Hoechst 33342 diluted in DPBS to assay plates by using Combi nL dispenser. The final concentration of Hoechst 33342 is 3.3 ug/mL.
10 Spin down plates on Eppendorf 5810 centrifuge at 1000 rpm for 1 minute. Then cover plates with aluminum plate seals.
11 Incubate plates for one hour prior to loading to PerkinElmer Opera, or store plates at 4 oC until imaging.
(1) Cell Growth Media and Cell Culture
NTR1-U2OS Osteosarcoma cells are maintained in MEM growth medium containing 10% Fetal Bovine Serum, 1% Penicillin Streptomycin solution, 1% L-Glutamine with 400 ug/mL G418 and 200ug/mL Zeocin at 37 oC with 5% CO2. Discard cells after 20 passages.
(2) Assay Media
MEM medium containing 2.5 % Fetal Bovine Serum, 1 % Penicillin Streptomycin solution, 1 % L-Glutamine with 400 ug/mL G418 and 200 ug/mL Zeocin.
(3) NTR1 peptide solution
1 Dissolve the powder in 50% Glycerol-H2O at 1 mM.
2 Store it as stock solution at 4 oC until just before use.
3 Dilute stock solution (1 mM) to the working conc. of 300 nM in DPBS.
*Prepare working solution (300 nM) just before use due to stability issue of the peptide.
(4) 6% Paraformaldehyde Solution
1 Place 2.4 L of MilliQ H2O in a container and raise pH to 11 using NaOH while stirring.
2 Add 180 g of Paraformaldehyde powder gently into solution.
3 Keep stirring at room temp until it is completely dissolved (this can take several hours).
4 Adjust pH to 7.4 using H2SO4 (not HCL).
5 Add MilliQ H2O to have the solution final volume 3L.
6 Store the solution at 4 oC. Protect from light. It can be stored up to a week.
(5) Hoechst solution
1 Dilute stock solution (10 mg/mL) to the working conc. of 5 ug/mL in DPBS.
J. Image Acquisition and Analysis:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 40x 0.6 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: 1) Beta-arrestin-GFP using 488 nm laser excitation and 540/75 nm emission filters, 2) DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
- 3 fields per well for Primary screen
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
- Threshold Adjustment: 1.5
- Minimum Nuclei distance: 7
- Nuclear Splitting Adjustment: 7
-Individual Threshold Adjustment: 0.4
- Minimum Nuclear Area: 70
- Minimum Nuclear Contrast: 0.1
- Cytoplasm Individual Threshold Adjustment: 0.1
- Spot Minimum Distance: 3
- Spot Peak Radius: 0
- Spot Reference Radius: 3
- Spot Minimum Contrast: 0.4
- Spot Minimum to Cell Intensity: 1
3) Metrics calculated from...
Cell Count ("CellCount"),
Nuclei Area ("NuclearArea"),
Integrated Intensity of the Nuclei ("TotNucIntensity"),
Average Intensity of the Nuclei ("AvgNucIntensity");
Average Intensity of the Cytoplasm ("AvgCytoIntensity"),
Average Intensity of the Cell ("AvgCellIntensity"),
Integrated Intensity of the Cell ("TotCellIntensity"),
Integrated Intensity of the Cytoplasm ("TotCytoIntensity"),
Integrated Intensity of the Detected Spots ("TotSpotIntensity"),
Ratio of the Integrated Spot to Integrated Cytoplasm Intensities ("RatioTotSpotIntensity2CellIntensity"),
Number of Spots per Cell ("AvgSpotPerCell"),
Average Area of Spots ("AvgSpotAreaPerCell").
Compounds with and EC50_Mean <= 20 uM are active.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)