qHTS for Inhibitors of ATXN expression
Spinocerebellar ataxia type 2 (SCA2) is a multisystem neurodegenerative disease caused by a dominantly-acting mutation leading to expansion of a polyQ domain in the ataxin-2 protein (ATXN2). SCA2 patients develop progressive ataxia and later lose function in other neuronal systems. Prominent parkinsonian signs develop in some patients. Similar to many neurodegenerative disorders, no symptomatic or disease-modifying treatments are known. ..more
BioActive Compounds: 3812
Spinocerebellar ataxia type 2 (SCA2) is a multisystem neurodegenerative disease caused by a dominantly-acting mutation leading to expansion of a polyQ domain in the ataxin-2 protein (ATXN2). SCA2 patients develop progressive ataxia and later lose function in other neuronal systems. Prominent parkinsonian signs develop in some patients. Similar to many neurodegenerative disorders, no symptomatic or disease-modifying treatments are known.
The primary objective of the proposed research is to identify compounds inhibiting ATXN2 expression and to test them for efficacy in SCA2 mouse models. To this end, we have developed a cell-based assay paired with an in vivo mouse model using identical luciferase expression constructs. This approach takes advantage of an ATXN2-luciferase transgene, with the luciferase gene flanked by the upstream and downstream portions of the ATXN2 gene. This primary assay was tested against the NIH Molecular Libraries Small Molecule Repository (MLSMR).
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: NS073009
Assay Submitter (PI): Daniel Scoles, University of Utah
4 microl of SH-SY5Y-ATXN2-FFLuc cell suspension (TBD cells/microL in phenol-red free DMEM) are dispensed into Greiner white solid bottom 1536-well assay plates, yielding a final well density of TBD cells/well. Plates are incubated for overnight at 37 degrees C in 5% humidified CO2 to allow cell attachment. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10nl slotted pins, V&P Scientific, San Diego, CA). After incubation at 37 degrees C in 5% humidified CO2 for 24 hrs, 1microl of GF-AFC (Gly-Phe-7-amino-4-trifluoromethyl coumarin, MP Biomedicals) at a final concentration of 25microM is then added and plates are incubated for 30 minutes, before being transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein single end-point fluorescence measurements are acquired using the 405 nm excitation/525 nm emission filter set to assess cell viability. Next, Steady-Glo luciferase substrate detection reagent (2microl) (Promega, Madison, WI) is added to each well and incubated for an additional 5 minutes. Luminescence is then measured on the ViewLux imager equipped with a clear emission filter using a 2 sec exposure.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)