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BioAssay: AID 1829

Broad Institute MLPCN Alpha-Synuclein 5'UTR - Activators

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 ..
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 Related BioAssays
AID: 1829
Data Source: Broad Institute (2023-activators)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-24
Modify Date: 2010-06-18
Related Experiments
Show more
AIDNameTypeComment
1814MLPCN Alpha-Synuclein 5'UTR - 5'-UTR binding - activatorsScreeningdepositor-specified cross reference: primary screening campaign of 303,811 compounds
1999Luminescence Cell-Based Dose Response HTS to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference
2002Luminescence Cell-Based Dose Response HTS to Identify Activators of Luciferase Translation or Activity in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Secondary screen of 2336 compounds
2003Luminescence Cell-Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Dose retest of 2336 compounds
2442Luminescence Cell-Based Dose Retest to Confirm Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Dose retest of 41 compounds
2446Luminescence Cell-Based Dose Response HTS to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Secondary screen of 41 compounds
2450Luminescence Cell-Based Dose Confimation to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Dose retest of 235 compounds
2453Luminescence Cell-Based Dose Response HTS to Identify Activators of Luciferase Translation or Activity in H4-C Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Secondary screen of 41 compounds
2456Luminescence Cell-Based Dose Response to Identify Activators of Luciferase Translation or Activity in H4-C Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Secondary screen of 235 compounds
2457Luminescence Cell-Based Dose Response to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference: Secondary screen of 235 compounds
2627Western Blot Cell-Based Dose Response to Identify Activators of Alpha-Synuclein Translation in H4 CellsConfirmatorydepositor-specified cross reference: Secondary screen of 1 compound
2670Luminescence Cell Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma CellsConfirmatorydepositor-specified cross reference
Description:
Broad Institute of MIT and Harvard, Cambridge MA

NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number 1-R21-NS-059434-01

Internal Project ID: 2023

Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region

Primary Collaborators:

Jack T. Rogers, Massachusetts General Hospital, Charlestown, MA, jtrogers@rics.bwh.harvard.edu

Probes:
ML163

Project Overview:
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.

Primary Assay Summary:

Name: 5'-UTR Binding

Internal Assay ID: 2023-01-BAssay Overview:

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 3.75 mM compound was added to the cell solutions (7.5 uM final [compound]). The calibration 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.


PubChem AID 1814

Number substances evaluated: 303857

Since the calibration control used acts as an inhibitor of this assay, activators were identified by selecting compounds with a negative PubChem_Activity_Score.

Active Compounds : Activity_Score <= -62.

Number of active compounds: 21690

Inconclusive Compounds: Activity_Score > -62 with at least one replicate activity score <=-62.

Number of inconclusive compounds: 5060
Comment
The following compounds [SID] were purchased or synthesized for this project:
87218802 87218803 87218805 87218894 87218904 87218911
87218912 87218913 87218914 87218915 87218916 87218917
87218918 87218919 87218920 87218921 87218922 87218923
87218924 87218925 87218926 87218927 87218928 87457165
87457166 87457167 89856174
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML163
PubChem Substance ID (SID) for probe 1: 87218802
PubChem Compound ID (CID) for probe 1: 1090779
Probe type for probe 1: Activator
IC50/EC50 (nM) for probe 1: 2000
Target for probe 1: alpha-synuclein 5'-UTR
Disease relevance for probe 1: Parkinson disease
Anti-target for probe 1: Prion 5'-UTR (H4-PRP)
Fold selectivity for probe 1: >100
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK55072/ (ID: 2477688)
Grant number for probe 1: NS059434-01
NCBI Book chapter title for probe 1: Identification of a small molecule that selectively activates alpha-synuclein translational expression
Additional Information
Grant Number: 1-R21-NS-059434-01

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