Screen for Chemicals that Extend Yeast Lifespan
There is now solid evidence for the existence of conserved pathways that regulate cell aging and senescence. These pathways may have evolved to allow eukaryotic cells and animals to remain reproductively viable for long periods during unfavorable environmental conditions. For example, lifespan extension by caloric restriction occurs in both yeast and rodents. Key elements of broadly conserved more ..
BioActive Compounds: 904
Depositor Specified Assays
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 existence of conserved pathways that regulate cell aging and senescence. These pathways may have evolved to allow eukaryotic cells and animals to remain reproductively viable for long periods during unfavorable environmental conditions. For example, lifespan extension by caloric restriction occurs in both yeast and rodents. Key elements of broadly conserved aging mechanisms, including the role of sirtuins in lifespan, were first discovered in Saccharomyces cerevisiae. This provides a strong rationale for the use of yeast as a genetic model system for studying aging.
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 replicative lifespan '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.
We used a genetically modified strain of S. cerevisiae in a high throughput replicative lifespan assay called the DeaD assay (1). Under permissive conditions, in a galactose-containing medium, these cells divide exponentially because all cells reproduce (mothers and daughters). Under restrictive conditions, in a glucose medium, the daughters show a great propensity to die, and the saturation point of the culture is limited by the lifespan of the mother cells rather than nutrient limitation. Nicotinamide is an inhibitor of the deacetylase Sir2p, and has been shown to reduce lifespan by both sirtuin-dependent and independent mechanisms (2, 3). Nicotinamide reduces the lifespan of the DeaD strain grown in restrictive medium without affecting growth under permissive conditions. To identify molecules that may extend replicative lifespan we devised a screen to search for compounds that reverse the lifespan shortening activity of nicotinamide.
The percent inhibition of nicotinamide lifespan shortening, i.e. percent reversal of nicotinamide effect on lifespan, was calculated using the optical density in control wells with cells treated with 1.5 mM nicotinamide as full lifespan shortening effect of nicotinamide (0% inhibition of nicotinamide), and wells with cells grown in medium without nicotinamide as an indicator of 100% inhibition. Library compounds were screened at 10 uM in the presence of 1.5 mM nicotinamide.
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.
2. Bitterman, K.J., Anderson, R.M., Cohen, H.Y., Latorre-Esteves, M., and D.A. Sinclair. (2002). Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast Sir2 and human SIRT1. J. Biol. Chem. 277, 45099-45107.
3. Tsuchiya, M., Dang, N., Kerr, E.O., Hu, D., Steffen, K.K., Oakes, J.A. , Kennedy, B.K. and M. Kaeberlein (2006) Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in yeast. Aging Cell. 5, 505-514.
Preparation of assay
1. Cells were streaked out on a YPGal agar plate and grown for 48 h at 30C.
2. 4 colonies were selected, 50 mL of YPGal medium in a flask was inoculated and grown at 30C with shaking O/N
4. OD600 was measured. The OD should be <0.7 for the cells to be in log phase.
5. The cells were centrifuged, washed once and resuspended in CSMM-D restrictive growth medium. OD600 was measured again. The culture was diluted to an OD600 of 0.002 in CSMM-D restrictive medium.
6. The culture was pre-incubated in a flask with shaking at 30C for 4 h. At the end of the pre-incubation, OD600 was measured for reference.
7. Nicotinamide (negative control), medium alone (positive control) and compounds in the presence of nicotinamide were plated with DMSO at 10 x concentration (final concentrations: nicotinamide 1.5 mM, compounds 10 uM, DMSO 0.25%) in 384-well plates: 5 uL/well.
8. The yeast was added to the plates: 45 uL/well. Plates were incubated at 30C in a humidified chamber.
9. After 48 h incubation, plates were shaken for 30 s and OD615 was read in an EnVision (PerkinElmer) multilabel plate reader.
10 g yeast extract
20 g peptone
900 mL water
Autoclave at 121C for 15 min
Add 100 mL sterile 20% (w/v) Galactose
CSMM-D (Complete Synthetic Minimal Medium-Dextrose) (restrictive) medium:
6.7 g yeast nitrogen base w/o amino acids
2.0 g Drop-out mix complete (DOC) (USBiological Cat. no. D9515)
100 mL 20% (w/v) dextrose
Water to 1.0 L
Possible artifacts in this assay include, but are not limited to, escape mutations occurring in cells allowing for extended growth in the presence of nicotinamide and restrictive medium as well as compounds that absorb light at 615 nm or precipitate.
Outcome: An activity threshold of >26% was calculated as greater than three standard deviations from the median compound inhibition. Therefore, compounds that exhibited >26% inhibition are defined as Active. Compounds that exhibited <=26% inhibition are defined as Inactive.
Because of the inherent error in all high throughput screens including the fallacy of over-interpreting single dose data, the following tiered scoring system has been implemented at SRMLSC. Compounds in this single dose primary screen were scored on a scale of 0-40, based on activity, with a scaled activity threshold of 11. Subsequent active compounds in confirmatory screens will be scored on a tier of 40-80, followed by resynthesized/analog active compounds screened in later stage probe development, which will fall in to the most reliable tier where actives will be scored from 80-100.
Data Table (Concise)