|Broad Institute MLPCN Alpha-Synuclein 5'UTR - Activators - BioAssay Summary
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 ..
Depositor Specified Assays
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
Jack T. Rogers, Massachusetts General Hospital, Charlestown, MA, firstname.lastname@example.org
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
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