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BioAssay: AID 2473

ELISA Cell-Based Dose Response to Identify Inhibitors of Alpha-Synuclein Translation in SH-SY5Y Cells

Jack T. Rogers, Massachusetts General Hospital, jtrogers@rics.bwh.harvard.edu, 617-726-8838, Charlestown, MA ..more
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AID: 2473
Data Source: Broad Institute (2023-04_INHIBITORS_DOSE-TITRATION_MLPCN-DRYPOWDER>)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-04
Hold-until Date: 2010-09-09
Modify Date: 2010-08-31

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
1813MLPCN Alpha-Synuclein 5'UTR - 5'-UTR binding - inhibitorsScreening depositor-specified cross reference: Primary HTS
1827Broad Institute MLPCN Alpha-Synuclein 5'UTR - InhibitorsSummary2 depositor-specified cross reference: Project Summary
1927FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Confirmatory same project related to Summary assay
1988Luminescence Cell-Based Dose Confimation HTS to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
1990Luminescence Cell-Based Dose Response HTS to Identify Inhibitors of Luciferase Translation or Activity in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
1994Luminescence Cell-Based Dose Response HTS to Identify Inhibitors of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2442Luminescence Cell-Based Dose Retest to Confirm Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2452Luminescence Cell-Based Dose Response to Identify Inhibitors of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2454Luminescence Cell-Based HTS Dose Confimation to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2458Luminescence Cell-Based Dose Response to Identify Inhibitors of Luciferase Translation or Activity in H4-C Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2460Luminescence Cell-Based Dose Confimation HTS to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells.Confirmatory same project related to Summary assay
2463Luminescence Cell-Based Dose Response HTS to Identify Inhibitors of Luciferase Translation or Activity in H4-C Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2468Luminescence Cell-Based Dose HTS Response to Identify Inhibitors of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2471Luminescence Cell-Based Dose-Confimation HTS to Identify Inhibitors of of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary assay
2484Western Blot Cell-Based Dose Response to Identify Inhibitors of Binding of Alpha-Synuclein Translation in H4 CellsConfirmatory same project related to Summary assay
2627Western Blot Cell-Based Dose Response to Identify Activators of Alpha-Synuclein Translation in H4 CellsConfirmatory same project related to Summary assay
2670Luminescence Cell Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatory same project related to Summary 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 (and laboratory where this assay was performed):
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:
In the ELISA orthogonal screen confirmation of alpha-synuclein tranlation inhibitors was sought. The ELISA used SH-SY5Y neuroglioblastoma cells that were not transfected. Compound was tested in 4-point dose in an effort to observe a dose-response effect.

Expected Outcome:
Inhibitors of alpha-synuclein translation should decrease alpha-synuclein protein levels.
Protocol
ELISA Protocol: (Invitrogen alpha-synuclein ELISA kit #KHB0061)
- Plate 6000 cells/well (50 uL) with phenol red free media
- Incubate 24 hours at 37C, 5% CO2, 95% humidity
- Pin 100 nL of compounds
- Incubate 48 hours at 37C, 5% CO2, 95% humidity
- Add 5uL Alamar blue solution per well, Incubate for 1 Hr at 370C, 5% CO2, 95% humidity
- Read for fluorescence (570nm excitation, 585nm emission)
- Remove media
- Add cell lysis solution and keep the plates on plate shaker for 1 Hr
- Remove 20 uL lysate and place in 500 uL epi tube
- Centrifuge the cell lysate at 13,000 rpm for 10 min
- Transfer 12 uL supernatant to 48 uL of standard diluent buffer (1:5)
- Add 50 uL of Standard curve and other samples on ELISA plate
- Wait 5 min.
- Pipette 50 uL of anti-alpha-Synuclein (Detection Antibody) solution, tap plate to mix
- Cover wells with plate cover and incubate for 3 hours at room temperature
- Wash wells 4 times with wash buffer
- Add 100 uL anti-rabbit IgG-HRP antibody solution
- Cover wells with the plate cover and incubate for 30 mins at RT
- Wash wells 4 times
- Add 100 uL of Stabilized Chromogen
- Keep sealed for 30 minutes at room temperature in the dark
- Add 100 uL of Stop Solution, tap plate to mix
- Read the absorbance of each well at 450 nm
Comment
Dose Response Data Analysis:
Three independent wells were evaluated for each compound concentration. Negative control condition (DMSO) was included.
Live Cell Correction Factor at dose was calculated as:
OD Alamar Blue at dose/ OD Alamar Blue at DMSO
ELISA values are the average of the independent wells for each compound concentration. The ELISA included a standard curve of recombinant alpha-synuclein (provided in the kit). The ELISA value is background normalized to the ELISA kit using the zero condition as described in the kit manual. ELISA values for each compound concentration are adjusted for live cells as follows:
OD ELISA at dose/ Live Cell Correction Factor at dose
The alpha-synuclein concentration values are extrapolated from the recombinant alpha-synuclein standard curve fit equation. Percent inhibition of alpha-synuclein was calculated based on the assumption that complete (100%) inhibition is attainable, as follows:
100 - (100 * (alpha-synuclein concentration at dose/ alpha-synuclein concentration at DMSO))

EC50 was determined to be the lowest dose that supported >= 50% of Maximum Observed Activity.
PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10 * Log(EC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
Dose Response is not observed.

Activity_Outcome = 2 (active)
Dose Response is observed with an EC50 < 0.2uM.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: SH-SY5Y
From ChEMBL:
Assay Type: Functional
Result Definitions
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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
3Max. Activitythe maximum activity value observedFloat%
4Activity at 100uM (100μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity at 6.25uM (6.25μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity at 0.195uM (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity at 0.006uM (0.006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
8Activity at 0uM (0μM**)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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