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BioAssay: AID 2471

Luminescence Cell-Based Dose-Confimation HTS to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells

Jack T. Rogers, Massachusetts General Hospital, jtrogers@rics.bwh.harvard.edu, 617-726-8838, Charlestown, MA ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(3)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(3)
 
 
Inactive(3)
 
 
AID: 2471
Data Source: Broad Institute (2023-01_INHIBITORS_DOSE-TITRATION_MLPCN-DRY-POWDER_SET2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-04
Hold-until Date: 2010-09-09
Modify Date: 2010-08-31

Data Table ( Complete ):           Active    All
BioActive Compounds: 3
Depositor Specified Assays
AIDNameTypeComment
1813MLPCN Alpha-Synuclein 5'UTR - 5'-UTR binding - inhibitorsscreeningPrimary HTS
1827Broad Institute MLPCN Alpha-Synuclein 5'UTR - InhibitorssummaryProject Summary
Description:
Assay Description:

Broad Institute MLPCN Alpha-Synuclein 5'UTR Project

Project ID: 2023

Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region

Primary Collaborators:

Jack T. Rogers, Massachusetts General Hospital, jtrogers@rics.bwh.harvard.edu, 617-726-8838, Charlestown, MA

Project Overview:

The goal of this project is to identify novel small molecule probes that inhibit alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a major new therapeutic target to retard the progression of Parkinson's disease (PD). The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully reduce alpha-synuclein expression levels as measured in this luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5'UTR stem-loop to confirm that probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein 5'UTR mRNA stem-loop fused to a luciferase reporter.

Assay Overview:

Compounds were assayed against H4 neuroblastoma cells transfected with a plasmid containing alpha-synuclein 5'UTR-luciferase gene fusion (Jack Rogers). Compounds that cause a reduction in luciferase expression will be further tested for proper target binding and specificity. Assays were conducted in 384-well format (Corning, 3570) with a 3000 cells/well coating density in 50 uL phenol red-free DMEM with 4.5 g/L D-glucose (Lonza, 12-917F) supplemented with 10 % FBS (Lonza, 14-503E), 200 uM L-glutamine (Lonza, 17-605E), 100 uM penstrep (Lonza, 17-602E) and 200 ug/mL geneticin (Invitrogen, 10131-027). Following overnight growth, 100 nL of compound was added to each well. Compounds were tested in 8-pt dose at 2-fold dilutions. Compound stock concentrations spanned 10.0 mM to 78.1 uM, while final compound concentrations in the assay plates spanned 20.0 uM to 156 nM. The positive control used in this assay was strophanthidine (Sigma, Cat.# S6626-250MG, Lot#038K1036), which was used in-plate (32 wells at 20 uM final [compound]). Cells were grown for 48 h, equilibrated to room temperature and 30 uL Steady-Glo (Promega, E2250) reagent was added. The plates were then incubated at room temperature for 30 min, and luciferase levels were measured.
Protocol
Assay Protocol:


Taken from 2023-01-A02-03 through 2023-01-A02-07

1) H4 neuroglioblastoma cells stably transfected with the alpha-synuclein 5'UTR-Luciferase gene fusion were grown to confluency in 35 mL complete media (DMEM with 4.5 g/L D-glucose (Lonza, 12-614Q) supplemented with 10% FBS (Lonza, 14-503E), 200 uM L-glutamine (Lonza, 17-605E), 100 uM penstrep (Lonza, 17-602E) and 200 ug/mL geneticin (Invitrogen, 10131-027)) in a T175 TC flask (BD Falcon, 353112) in a TC incubator (37 C, 95 % humidity, 5 % CO2) (doubling time = 24 h).
2) Cells were harvested by washing the monolayer quickly with 5 mL trypsin/EDTA (1X, Cellgro, 25-053-Cl), aspirating, then adding 5 mL trypsin/EDTA and incubating for 5 min at 37C, 95% humidity, 5% CO2. Five mL of complete media was then added and mixed by pipetting up and down. This 10 mL of cell solution was then aspirated and added to a conical tube and cells were pelleted for 3 min at 1000 rpm in a swinging bucket centrifuge. The media was aspirated and the cell pellet was resuspended in 10 mL fresh complete media. Cells were then counted and expanded (see below).
3) Cells were expanded by plating 1x10^6 cells in 35 mL complete media per T175 flask (generally used 20xT175 flasks per 200 assay plate run) and allowing 72 h for cells to grow to confluency.
4) Cells were harvested as above, but were resuspended in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose (Lonza, 12-917F) supplemented with 10 % FBS (Lonza, 14-503E), 200 uM L-glutamine (Lonza, 17-605E), 100 uM penstrep (Lonza, 17-602E) and 200 ug/mL geneticin (Invitrogen, 10131-027)) and then were diluted to 60,000 cells/mL in phenol-red free complete media prewarmed to 37 C in 1 L sterile plastic bottles.
5) Cells were plated into 384-well plates (batch size = 200-220 assay plates) at 3,000 cells/well in 50 uL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser and a sterilized dispensing cassette and stir bar, in a TC hood.
6) Assay plates were loaded into 22 slot holders which were then placed into an online Liconic (STX 2201C) incubator set to 37 C, 95 % humidity, 5 % CO2, and were incubated overnight.
7) Screening was performed using an open system (HiRes Biosolutions). Each run was initiated in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system. Replicate assay plates were delidded and then pinned with 100 nL 3.75 uM compound (final concentration = 7.5 uM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions).
8) Plates with compound were then relidded and returned to the Liconic incubator (STX 2201C) and incubated for 48 h at 37 C, 95 % humidity, 5 % CO2.
9) Plates were then individually moved to a room temperature Liconic Carousel (LPX 220) and were allowed to temperature equilibrate for 30 min.
10) Plates were then delidded and moved to a MultiDrop Combi liquid dispenser where 30 uL of 0.5X Steady-Glo (Promega, E2250, lot 273368) was added (Steady-Glo mainted at 4 C and warmed to room temperature using a Combi cassette with a 6 ft input line submerged in a room temperature water bath).
11) Plates were then relidded and returned to the Liconic Carousel and incubated at room temperature for 30 min.
12) Plates were then delidded and moved to an Envision plate reader (Perkin Elmer) and Luminescence values were collected using with 100 ms read time per well.
13) Plates were then relidded and discarded.
Comment
Data Analysis:

Dose Response Data Analysis:
32 Negative control (DMSO) and 32 positive control (Strophanthidine) wells were included on each plate.

All wells tested were background subtracted using the median of the negative control wells on the same plate and scaled using the median value of the positive control wells on the same plate. Plate pattern correction was not used.

IC50 values were calculated using the Smart Fit or Robust Fit strategy of Genedata
Screener Condoseo (v7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Standard Errorthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7Num. Pointsthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9Activity at 0.0000380uM (3.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.0000750uM (7.5e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.000150uM (0.00015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.000300uM (0.0003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 0.000600uM (0.0006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat
14Activity at 0.00120uM (0.0012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 0.00235uM (0.00235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 0.00500uM (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity at 0.01000uM (0.01μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity at 0.0195uM (0.0195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity at 0.0380uM (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity at 0.0800uM (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity at 0.160uM (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity at 0.300uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity at 0.600uM (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity at 1.20uM (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity at 2.60uM (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity at 5.00uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity at 10.00uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity at 19.5uM (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1-R21-NS-059434-01

Data Table (Concise)
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