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BioAssay: AID 1994

Luminescence Cell-Based Dose Response HTS to Identify Inhibitors of 5'UTR Stem-Loop Driven Prion Protein 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(2193)
 
 
Active(1193)
 
 
Inactive(853)
 
 
Inconclusive(147)
 
 
 Tested Substances
 Tested Substances
All(2193)
 
 
Active(1193)
 
 
Inactive(853)
 
 
Inconclusive(147)
 
 
 Related BioAssays
 Related BioAssays
AID: 1994
Data Source: Broad Institute (2023-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-19
Modify Date: 2009-12-14

Data Table ( Complete ):           Active    All
BioActive Compounds: 1193
Depositor Specified Assays
AIDNameTypeComment
1813MLPCN Alpha-Synuclein 5'UTR - 5'-UTR binding - inhibitorsscreeningPrimary HTS
1827Broad Institute MLPCN Alpha-Synuclein 5'UTR - InhibitorssummaryProject Summary
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, which is fused to a luciferase reporter.

Assay Overview:
Compounds were assayed against H4 neuroblastoma cells transfected with a dicistronic plasmid containing a viral internal ribosome entry site stem-loop sequence upstream of the 5'UTR stem-loop sequence from Prion protein (PRP) fused to the luciferase gene (Jack Rogers). Compounds that cause a reduction in luciferase expression will be compared to their activity in the primary screening line to eliminate false positive hits due to non-specific RNA stem-loop binding. 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 Pen-Strep (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 6.25 mM to 24 uM, while final compound concentrations in the assay plates spanned 12.5 uM to 48.8 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 for 30 min 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
1) H4 neuroglioblastoma cells stably transfected with the Prion protein (PRP) 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 Pen-Strep (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 2x10^6 cells in 50 mL complete media per T225 flask 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 Pen-Strep(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 = 100 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 offline Liconic (STX 2201C) incubator set to 37 C, 95 % humidity, 5 % CO2, and were incubated overnight.
7) Screening was performed using walk-up instrumentation. Each run was initiated in CBIP (Broad Chemical Biology Informatics Platform). In groups of 22, assay plates were delidded and stacked. They were then individually pinned with 100 nL compounds, which were tested in 8-pt dose at 2-fold dilutions. Compound stock concentrations spanned 6.25 mM to 24 uM, while final compound concentrations in the assay plates spanned 12.5 uM to 48.8 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]).
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 moved in sets of 22 to room temperature 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 maintained 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 were allowed to incubate 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 discarded.
Comment
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 controls 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 strategy of Genedata
Screener Condoseo (v6.0.1). 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)
Constant fit curve activity is >30% but <70%
or
Constant fit curve is labeled as invalid due to large fit error
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 0.10uM (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.19uM (0.19μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.75uM (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 0.80uM (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 1.50uM (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 1.60uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 3.00uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity at 6.00uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity at 12.00uM (12μM**)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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