Broad Institute MLPCN Alpha-Synuclein 5'UTR - Inhibitors
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), more ..
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 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.
Primary Assay Summary:
Name: 5'-UTR Binding
Internal Assay ID: 2023-01
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.
PubChem AID 1813
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.
62 represents a score that is 4x the standard deviation of all negative control (DMSO) samples in the screen.
Number of active compounds: 2498
Inconclusive Compounds: Activity_Score < 62 with at least one replicate activity score >=62.
Number of inconclusive compounds: 481
The following compounds [SID] were purchased or synthesized for this project:
87218804 87218892 87218893 87218896 87218898 87218900
87218901 87218902 87218903 87218905 87218906 87218907
87218908 87218909 87218910 87457149 87457152 87556803
87556804 87556806 87556807 87556808 87556809
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML150
PubChem Substance ID (SID) for probe 1: 87218804
PubChem Compound ID (CID) for probe 1: 1517919
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 1800
Target for probe 1: alpha-synuclein 5'-UTR
Disease relevance for probe 1: Parkinson disease
Anti-target for probe 1: Prion 5'-UTR
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK55070/ (ID: 2477392)
Grant number for probe 1: NS059434-01
NCBI Book chapter title for probe 1: Identification of a small molecule that selectively inhibits alpha-synuclein translational expression
Data Table (Concise)