SAR analysis of small molecule NTR1 agonists in a image based NTR2 selectivity assay set 2
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: 16
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. In the screen, "Image-Based HTS for Selective Agonists for NTR1"(AID 493036), we have identified numerous hits that may be selective agonists of the NTR1 receptor using a primary assay based upon the ability of a -arrestin fluorescent reporter to directly recognize the activated state of the NTR1.
The goal of this assay is to assess the selectivity of NTR1 hits from "Image-Based HTS for Selective Agonists for NTR1", (AID 493036) and to study the structure-activity relationship on analogs of the confirmed hits against the neurotensin receptor 2 (NTR2). This is accomplished via the same methodology used in the NTR1 primary assay, the detection of beta-arrestin fluorescent reporter evident upon NTR2 activation. Compounds are either acquired from commercial sources or synthesized internally.
Biology of neurotensin: revisited study. Katsanos GS, Anogianaki A, Castellani ML, Ciampoli C, De Amicis D, Orso C, Pollice R, Vecchiet J, Tete S, Salini V, Caraffa A, Patruno A, Shaik YB, Kempuraj D, Doyle R, Antinolfi PL, Cerulli G, Conti CM, Fulcheri M, Neri G, Sabatino G. Int J Immunopathol Pharmacol. 2008 Apr-Jun;21(2):255-9.
Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor. D Gully, M Canton, R Boigegrain, F Jeanjean, J C Molimard, M Poncelet, C Gueudet, M Heaulme, R Leyris, A Brouard, et al. Proc Natl Acad Sci U S A. 1993 January 1; 90(1): 65-69.
Neurotensin receptor antagonists and therapeutical perspectives. Gully D, Maffrand JP, Soubrie P, Rostene W, Kitabgi P, Le Fur G. Therapie. 1995 Jan-Feb;50(1):5-7.
Neuropharmacological profile of non-peptide neurotensin antagonists. Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrie P, Le Fur G, Maffrand JP. Fundam Clin Pharmacol. 1995;9(6):513-21. Review.
Use of nonpeptide antagonists to explore the physiological roles of neurotensin. Focus on brain neurotensin/dopamine interactions. Rostene W, Azzi M, Boudin H, Lepee I, Souaze F, Mendez-Ubach M, Betancur C, Gully D. Ann N Y Acad Sci. 1997 Apr 24;814:125-41. Review.
A. Brief Description of the Assay:
The goal of this assay is to assess the selectivity in dose response of the NTR1 agonist hits against the neurotensin receptor 2 (NTR2). Compounds that are active in this assay against the related GPCR, NTR2, are believed to be non-selective for NTR1. The compounds' activity may still be specific to the neurotensin family of receptors.
Description Source Cat
NTR2-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
NTR2-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 and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pEC50 - 3),
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)