Luminescence Cell Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells
The goal of this project is to identify novel small molecule probes that increase alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a novel probe for alpha-synuclein translational expression. The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction more ..
BioActive Compounds: 4
Project ID: 2023
alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region
Jack T. Rogers, Mass General Hospital, email@example.com, 617-726-8838, Charlestown, MA
The goal of this project is to identify novel small molecule probes that increase alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a novel probe for alpha-synuclein translational expression. 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. Increased levels of alpha-synuclein have been directly linked to Parkinson's disease. Probes that can successfully increase 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.
Compounds were assayed against H4 neuroblastoma cells transfected with a plasmid containing alpha-synuclein 5'UTR-luciferase gene fusion (Jack Rogers). Compounds that cause an increase 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 6.25 mM to 24 uM, while final compound concentrations in the assay plates spanned 12.5 uM to 48.8 nM. 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.
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.
Dose Response Data Analysis:
Negative control (NC) wells and inhibitor controls (IC) were included on each plate.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate IC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
Active compounds resulted in increased signal relative to NC. (i.e. >0)
No plate pattern correction algorithm was applied.
IC50 values were calculated using the Smart Fit or Robust Fit strategy of Genedata Screener Condoseo (v7.0.3), with "automatic parameter fixing" deselected and the "maximal span" set to 20,000.
EC50 values were extrapolated up to 1 log
over the highest tested concentration.
Inactive compounds = 0
Active compounds = -10*Log(EC50)
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%
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: H4
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)