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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Depositor Specified Assays
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| AID | Name | Type | Comment |
|---|---|---|---|
| 1814 | MLPCN Alpha-Synuclein 5'UTR - 5'-UTR binding - activators | screening | primary screening campaign of 303,811 compounds |
| 2003 | Luminescence Cell-Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells | confirmatory | Dose retest of 2336 compounds |
| 2450 | Luminescence Cell-Based Dose Confimation to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells | confirmatory | Dose retest of 235 compounds |
| 2442 | Luminescence Cell-Based Dose Retest to Confirm Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells | confirmatory | Dose retest of 41 compounds |
| 2446 | Luminescence Cell-Based Dose Response HTS to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma Cells | confirmatory | Secondary screen of 41 compounds |
| 2457 | Luminescence Cell-Based Dose Response to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4-C Neuroglioblastoma Cells | confirmatory | Secondary screen of 235 compounds |
| 2453 | Luminescence Cell-Based Dose Response HTS to Identify Activators of Luciferase Translation or Activity in H4-C Neuroglioblastoma Cells | confirmatory | Secondary screen of 41 compounds |
| 2456 | Luminescence Cell-Based Dose Response to Identify Activators of Luciferase Translation or Activity in H4-C Neuroglioblastoma Cells | confirmatory | Secondary screen of 235 compounds |
| 2627 | Western Blot Cell-Based Dose Response to Identify Activators of Alpha-Synuclein Translation in H4 Cells | confirmatory | Secondary screen of 1 compound |
| 2002 | Luminescence Cell-Based Dose Response HTS to Identify Activators of Luciferase Translation or Activity in H4 Neuroglioblastoma Cells | confirmatory | Secondary screen of 2336 compounds |
| 1999 | Luminescence Cell-Based Dose Response HTS to Identify Activators of 5'UTR Stem-Loop Driven Prion Protein mRNA Translation in H4 Neuroglioblastoma Cells | confirmatory | |
| 2670 | Luminescence Cell Based Dose Confimation HTS to Identify Activators of 5'UTR Stem-Loop Driven Alpha-Synuclein mRNA Translation in H4 Neuroglioblastoma Cells | confirmatory |
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
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
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
Additional Information
Grant Number: 1-R21-NS-059434-01
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