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BioAssay: AID 2484

Western Blot Cell-Based Dose Response to Identify Inhibitors of Binding of Alpha-Synuclein Translation in H4 Cells

Jack T. Rogers, Massachusetts General Hospital, jtrogers@rics.bwh.harvard.edu, 617-726-8838, Charlestown, MA ..more
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AID: 2484
Data Source: Broad Institute (2023-05_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: 2010-03-05
Hold-until Date: 2010-09-09
Modify Date: 2010-08-31

Data Table ( Complete ):           Active    All
BioActive Compound: 1
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 (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 Western blot orthogonal screen confirmation of alpha-synuclein translation inhibitors was sought. The Western used H4 neuroblastoma 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 without affecting actin levels.
Protocol
Cell Culture and Preparation of Lysates:

1. Human H4 neuroglioblastoma cells were cultured in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD). Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10, 100 micromolar) for 48 hours.

2. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).

Western Blot:

1. Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon).

2. The blots were developed using chemiluminescence (PIERCE) and visualized with a PhosphoImager (BioRad, Hercules, CA), and bands were quantified using QuantityOne software (BioRad).
Comment
Dose Response Data Analysis:
Negative control condition (DMSO) was included.
SNCA Correction Factor was calculated as:
Density SNCA at neg/ Density Actin at neg

Percent inhibition of SNCA expression was calculated based on the assumption that complete (100% ) inhibition is attainable, as follows:
100 - ((100/Correction Factor) * Density SNCA at dose/ Density Actin at dose)

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 < 10uM.
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 10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity at 1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity at 0.1uM (0.1μM**)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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