Yeast Lifespan Shortening Chemical Screening, Permissive Growth Control - Pilot Screen
There is now solid evidence for the early evolution of conserved pathways for aging. These pathways may allow eukaryotic cells and animals to postpone reproduction in unfavorable environmental conditions. Key elements of public cellular mechanisms that extend or shorten lifespan, including the role of sirtuins in lifespan, were first discovered in Saccharomyces cerevisiae, which provides a strong rationale for the use of yeast as a model genetic system for studying aging. For example, lifespan extension by caloric restriction occurs in both yeast and rodents. ..more
BioActive Compounds: 23
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. David S. Goldfarb, University of Rochester
Award: R03 MH076395-01
There is now solid evidence for the early evolution of conserved pathways for aging. These pathways may allow eukaryotic cells and animals to postpone reproduction in unfavorable environmental conditions. Key elements of public cellular mechanisms that extend or shorten lifespan, including the role of sirtuins in lifespan, were first discovered in Saccharomyces cerevisiae, which provides a strong rationale for the use of yeast as a model genetic system for studying aging. For example, lifespan extension by caloric restriction occurs in both yeast and rodents.
Yeast replicative lifespan is the number of times a mother cell replicates before she senesces and dies. The replicative lifespan of a yeast strain is described by the mean or median lifespan of a cohort of mother cells, which can vary widely among laboratory strains, but is normally between 20-25 generations. The clock for daughters is generally reset to zero although daughters of older mothers, which replicate more slowly, have reduced lifespans. The genetic program(s) that sets the clock, and the cellular mechanisms that respond to environmental cues to extend lifespan, such as caloric restriction, are poorly understood.
Here we use a genetically modified version of a high throughput replicative lifespan assay called the DeaD assay (1). Under permissive conditions, in galactose-containing medium, the culture divides exponentially. Under restrictive conditions, in glucose medium, the daughters show a great propensity to die, and the saturation point of the culture is limited by the lifespan of the of mother cells rather than nutrient limitation. Compounds that reduce growth under permissive conditions are likely growth inhibitory or cytotoxic, and not lifespan shortening per se. This serves as a control screen to sort out false positives from a primary screen under restrictive medium. Compounds that reduce growth under restrictive conditions, but have no effect on growth under permissive conditions are candidates for targeting lifespan regulatory pathways. Percent inhibition of growth was calculated by using the optical density in control wells with untreated cells as full growth/lifespan (0% inhibition) and wells treated with 10 ug/mL amphotericin B as complete inhibition of growth (100% inhibition). Library compounds were screened at 10 uM.
1. Jarolim, S., Millen, J., Heeren, G., Laun, P., Goldfarb, D.S. and M. Breitenbach. (2004). A novel assay for replicative lifespan in Saccharomyces cerevisiae. FEMS Yeast Res. 5, 169-177.
Preparation of assay
1. Thaw stock vial and wash once in SC Raff/Gal permissive media.
2. Resuspend pellet in SC Raff/Gal permissive media to provide an OD600 of 0.002.
3. Grow O/N with shaking at 30C
4. Measure OD600. The OD should be <0.6 for the cells to be in log phase.
5. Spin down and resuspend in CSMM-D restrictive growth medium. Measure OD600. Dilute (at least 200-fold) to a starting OD600 of 0.001 in SC Raff/Gal permissive medium.
6. Grow in flask with shaking at 30C for 4 h.
7. Plate compounds, control drug or medium with DMSO at 10 x concentration (final DMSO concentration: 0.25%, final compound concentration 10 uM) in 384-well plates (Corning cat. no. 3701). 5 uL/well
8. Plate the yeast into the plates: 45 uL/well. Incubate at 30C in a humidified chamber.
9. After 24 h incubation, shake plate 30 s and read OD615 in EnVision.
SC Raff/Gal (permissive) medium:
6.7 g yeast nitrogen base w/o amino acids (Sigma cat. no. Y 0626)
10 mL amino acid mix
100 mL 20% (w/v) raffinose
5 mL 20% (w/v) galactose
Water to 1.0 L
CSMM-D (Complete Synthetic Minimal Medium-Dextrose) (restrictive) medium:
6.7 g yeast nitrogen base w/o amino acids
10 mL amino acid mix
100 mL 20% (w/v) dextrose
Water to 1.0 L
Amino acid mix (in water):
Adenine, 2.0 g/L
Arginine, 2.0 g/L
Aspartic acid, 10.0 g/L
Glutamic acid, 10.0 g/L
Histidine, 2.0 g/L
Isoleucine, 3.0 g/L
Leucine, 6.0 g/L
Lysine, 3.0 g/L
Methionine, 2.0 g/L
Phenylalanine, 5.0 g/L
Serine, 40.0 g/L
Threonine, 20.0 g/L
Tryptophan, 2.0 g/L
Tyrosine, 3.0 g/L
Uracil, 2.0 g/L
Valine, 15.0 g/L
Possible artifacts in this assay include, but are not limited to, compounds that absorb light at 615 nm or precipitate.
Outcome: An activity threshold of >25% was calculated as greater than three standard deviations from the mean compound inhibition. Compounds that exhibited >25% inhibition in both assay replicates were defined as Active. Compounds that exhibited >25% inhibition in only one of the replicates were defined as Inconclusive. Compounds that exhibited <=25% inhibition in both replicates were defined as Inactive.
Because of the inherent error in all high throughput screens, including the inherent error in single dose data, compounds that were active in this screen were assigned a score of 100. All other compounds were assigned a score of 0.
Data Table (Concise)