Luminescence Cell-Based HTS Dose Confimation to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells
Jack T. Rogers, Massachusetts General Hospital, firstname.lastname@example.org, 617-726-8838, Charlestown, MA ..more
BioActive Compounds: 153
Depositor Specified Assays
Broad Institute MLPCN Alpha-Synuclein 5'UTR Project
Project ID: 2023
Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region
Jack T. Rogers, Massachusetts General Hospital, email@example.com, 617-726-8838, Charlestown, MA
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.
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 3.75 mM compound was added to the cell solutions (7.5 uM final [compound]). The positive control used in this assay was strophanthidine (Sigma, Cat.# S6626-250MG, Lot#038K1036), which was used in dose (12-point, 2-fold dilution, from 20 uM final [compound]) and was incorporated at the beginning and end of each days run. 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 pre-warmed 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 re-lidded 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 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 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.
HTS Data Analysis:
Negative control wells (DMSO) were included on every plate.
The compound Strophanthidine which inhibits the luciferase signal was used as a positive control for this assay. Two plates containing the positive control Strophanthidine at various doses were included in every assay run. In order to provide a consistent calibration for all runs, we defined the positive control value to be the value observed when the positive control was present at 20 uM.
The PubChem_Activity_Score was derived using the follow procedure:
1. A background-subtracted value was calculated for each well by subtracting the median value of the negative control wells on each plate from the value of each well on that plate.
2. Systematic plate effects were corrected for each run. A plate effect correction matrix was derived by calculating the median value of all non-positive-control wells for each well location (e.g. C07) across all the plates in a run. This correction matrix was then smoothed using an inverse-distance weighted median filter and subtracted from all wells on the plates in the same run resulting in a background-subtracted, plate-effect-corrected value for each well.
3. An activity score was derived for each well by dividing the background-subtracted, plate-effect-corrected value for each well by the median of the background-subtracted, plate-effect-corrected value of the positive control wells in the same run and multiplying the resulting fraction by 100.
4. The final PubChem_Activity_Score represents the mean of all valid replicate activity scores obtained. This score can be viewed as a measure of per cent inhibition of the assay relative to the positive control used for this screen.
The PubChem_Activity_Outcome class was assigned as described below:
Activity_Outcome = 1 (inactive)
PubChem_Activity_Score <62 and all replicate activity scores <62.
Activity_Outcome = 2 (active)
PubChem_Activity_Score >=62. 62 represents a score that is 4x the standard deviation of all negative control (DMSO) samples in the screen.
Activity_Outcome = 3 (inconclusive)
PubChem_Activity_Score < 62 with one of two replicate activity scores >=62.
Activity_Outcome = 4
* Activity Concentration. ** Test Concentration.
Data Table (Concise)