Screen for compounds that inhibit protein aggregation formed by mutant SOD1 (GFPSOD1)
Amyotrophic Lateral Sclerosis is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cortex, brain stem and spinal cord. 90% of the cases are sporadic and the rest is familial cases. About 10-20% of FALS cases are caused by missense mutation in the SOD1 gene, which encodes the cytoplasmic Cu, Zn superoxide dismutase. Mice expressing human FALS-linked SOD1 gene more ..
Amyotrophic Lateral Sclerosis is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cortex, brain stem and spinal cord. 90% of the cases are sporadic and the rest is familial cases. About 10-20% of FALS cases are caused by missense mutation in the SOD1 gene, which encodes the cytoplasmic Cu, Zn superoxide dismutase. Mice expressing human FALS-linked SOD1 gene develop age dependent ALS-like disorder characterized by degeneration of spinal cord motor neurons. Another prominent phenotype in these mice is the presence of neurofilament-rich cytoplasmic inclusions resembling those found in human sporadic and familial ALS. Increasing evidences suggest that FALS pathology results from toxic gain of function in SOD1. Several hypotheses have put forward to explain the biochemical nature of this toxic gain of function. One of them argues that the SOD1 toxicity results from the tendency of mutant SOD1 to form aggregates in the cytoplasm. To prove this hypothesis, we generated replication deficient adenovirus expressing wild type and mutant form of SOD1 fused to GFP. Upon infecting COS cells with these virus, mSOD-GFP fusion protein forms cytoplasmic aggregates but not wSOD-GFP fusion protein. We have developed a protocol for an image-based assay to screen for small chemical compounds that can inhibit the aggregate formation. Hopefully one can use these compounds to understand the biochemical nature of the toxicity of mutant SOD1.
Screen: Cos1 cells were grown in growth media in 150mm plate to 80% confluency. The day before the assay, detach cells using Trypsin/EDTA. Mix the cells with the virus (expressing G85R-GFP fusion protein). The ratio of cell to virus varies according to the titer of the virus. Plate the mixture in growth media in 384-well plate (Costar) at 4000 cells/well, 30 _l/well. Incubate the plates at 37oC overnight (16 hours). Pin transfer chemical library and vortex the plate for 20 seconds immediately after the pin transfer. Two different size pins were used 50nl and 40nl. The final concentrations of compounds used in the assay are 30_M and 10_M. And the assay is done in duplicates with both concentrations. Incubate the plates at 37oC for 5 hours. Add 10_l/well of proteasome inhibitor ALLN (ALLN #208719) to final concentration of 10_g/ml with a liquid handling system (Multidrop 384, Labsystems). Incubate overnight at 37oC. Aspirate the media from the plates. Fix cells with 4% paraformaldehyde for 20 minutes at room temperature. Wash with PBS 3x5 minutes. Stain cells with DAPI for 5 min. Wash again with PBS 3x5 minutes. The fluorescent images are collected by an autoscope. Two different views with 10_M distance were collected for each well.
Cell Lines: COS1 cells were infected with replication deficient adenovirus expressing wild type SOD1-GFP fusion protein or G85R SOD1-GFP fusion protein.
Growth Medium: COS1 cells are grown in DMEM with 10% FBS and 1% penicillin and streptomycin.
Assay Medium: The same growth media is used for the assay.
Assay Buffer: Phosphate Buffer Saline (PBS).
Assay Reagent: Cells were fixed in 4% paraformaldehyde and washed with PBS.
Data Analysis Images collected by autoscope are reviewed by eye. And the positive hits are noted and the original well is examined under microscope directly to be confirmed.
Dilution Series: The assay was performed at 30_M and 10_M. Dilution series are: 30_M, 15_M, 10_M, 5_M, 2_M and 1_M. The dilutions are done manually with a multichannel pipette.
Assay Type: mammalian-cell based
Number Replicates: 4
Assay Readout: visual detection
Detection Instrument Multidrop 384, LabsystemsVMP 384 pinhead attached to SEIKO robot arms on a platform designed by Newport Autoscope.
Liquid Handling Robot Multidrop 384, Labsystems
Liquid Handling Robot The chemical compounds are transferred using VMP 384 pinhead attached to a robot arm made by SEIKO Instrument.
Concentration Primary Screen: (uM) 10
Concentration Primary Screen: (uM) 30
Secondary screen is to examine the effect of hit compounds on microtubule morphology by immunofluorescence analysis using antibodies against - tubulin. Two different concentrations with duplicates were tested.
PI Name Qing Liu
Affiliation Institute of Chemistry and Cell Biology/Harvard Medical School
Investigator David Chalmers
A Note about the Activity Matrix Rankings:
The data for each assay has been converted into a numerical ranking of all 1040 compounds for that assay. Thus, a compound with a rank of 1 was the top scoring compound in that assay and a compound with a rank of 1040 was the lowest scoring compound in that assay. Compounds with equal scoring were given equal rank.
PubChem scores were derived as (1040-NINDS activity rank). Substances with reported EC50 values were considered active.
Data Table (Concise)