Yeast cell-based screen for compounds that suppress polyglutamine aggregation in vivo (75Q-TubH)
Huntington's Disease is a late onset neurodegenerative disease caused by expression of an expanded polyglutamine tract. A great deal of experimental evidence indicates that expanded polyglutamine tracts have an enhanced propensity for aggregation, and that this property is related to expanded polylgutamine toxicity. It is therefore of interest to discover compounds that can influence more ..
Huntington's Disease is a late onset neurodegenerative disease caused by expression of an expanded polyglutamine tract. A great deal of experimental evidence indicates that expanded polyglutamine tracts have an enhanced propensity for aggregation, and that this property is related to expanded polylgutamine toxicity. It is therefore of interest to discover compounds that can influence polyglutamine aggregation. We have observed that expression of an expanded polyglutamine-tub1 fusion protein is toxic in a yeast strain bearing a temperature-sensitive tub1 allele. This toxicity is associated with, and likely caused by, polyglutamine aggregation. Therefore, suppression of this toxicity (as measured by suppression of growth inhibition) is used to screen for compounds that suppress polyglutamine aggregation.
Cell Lines Saccharomyces cerevisiae strain DBY6745 was stably transformed with the CEN plasmid, 75Q-Tub1 that contains the HIS3 selectable marker. These plasmids contain 75 polyglutamines fused to the TUB1 ORF under a GAL promoter. This cell line is referred to here as 75Q-TubH.
75Q-TubH strain was grown in synthetic media lacking histidine respectively (to select for plasmids) supplemented with 2% raffinose.
Assay Buffer Not Applicable
Assay Reagent NONE
Screen Screening - incubation of cells with test compounds Cells were grown in selective media containing raffinose and grown to mid-log phase. Cells were then diluted to an OD660 of ~0.005 in selective media containing raffinose, galactose and 0.5% DMSO. 200 micro-l of cells were robotically aliquoted into 96-well assay plates (Corning Costar plates, N3598). 1 mkL of each compound from the NINDS library was then added to reach a final concentration of 50 mM in 1% DMSO (diluted with assay medium by 200x from the 10 mM stock solution in DMSO). The cells were incubated with the test compounds at 30 degrees C for ~70 hours. A Beckman Biomek 2000 was used to aliquot cells and compounds. An initial absorbance reading was performed. After incubation, the assay plates were shaken on a Fisher Vortex Genie 2 and immediately transferred to an absorbance plate reader. The plate was shaken an additional one second prior to reading. All plate readings were performed using a Wallac Victor 2 plater reader.
Data Analysis The initial plate reading was subtracted from all absorbance readings. The corrected absorbance from each compound-treated well was divided by the mean of all 13 96-well plates, columns 2-11. Readings from the negative controls (columns 1 and 12) were discarded due to sporadic evaporation of media from the vertical edges of the plates which results in inaccurately high absorbance readings. Each compound library plate was tested once with each of the two cell lines.
Dilution Series 34 compounds were selected for retesting in a 4-point dilution series with a maximum concentration of 100 uM. These dilutions were done by hand. Once created, the assay was performed as described in the screening section. Of these 34 retests, 11 were taken through a second retesting and of those, 2 were retested a third time. Concentrations required to achieve 50% of the maximally achievable effect for each compound (EC50) were calculated.
Detection Reagent NONE
Assay Type yeast-based
Number Replicates 1
Assay Readout absorbance
Detection Instrument Wallac Victor 2
Liquid Handling Robot Beckman Biomek 2000
Concentration Primary Screen (#M) 50
Number Negative Controls 208
Negative Control Mean 0.0
Negative Control Standard Deviation 0.0
Number Positive Controls 16
Positive Control Mean 0.096
Positive Control Standard Deviation 0.013
Mean of negative controls not reliable. No negative control standard deviation available. MEAN_OF_POSITIVE_CONTROLS: 75Q-Tub1H = 0.096 STANDARD_DEVIATION_OF_POSITIVE_CONTROLS: 75Q-Tub1H = 0.013.
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.
PI Name Stanley Fields
Affiliation HHMI, Departments of Medicine and Genome Sciences, University of Washington
Investigator Russell S.Lo
PI Name Robert E. Hughes
Affiliation Department of Medicine, University of Washington
Data Table (Concise)