Screen for Chemicals that Shorten 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: 710
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 have used a genetically modified strain of S. cerevisiae in a high throughput replicative lifespan assay called the DeaD assay (Jarolim et al, 2004). 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-containing 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 histone deacetylase Sir2p, and has been shown to reduce lifespan by both sirtuin-dependent and independent mechanisms (Bitterman et al 2002; Tsuchiya et al, 2006). Nicotinamide reduces the lifespan of the DeaD strain (BB579) grown in restrictive medium without affecting growth under permissive conditions. Therefore, compounds that reduce growth under restrictive conditions, but have no or little effect on growth under the control permissive condition are candidates for targeting lifespan regulatory pathways. In this single dose screen the yeast was grown in the plates under restrictive conditions. Compounds that negatively affect growth will be further tested in dose response assays under both restrictive and permissive conditions for identification of those that are potentially inhibiting replicative lifespan.
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.
Preparation of assay
1. Cells (DeaD strain BB579) 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. CSMM-D medium alone (negative control), amphotericin B (positive control) and compounds were plated with DMSO at 10 x concentration (final concentrations: amphotericin B 10 ug/mL, 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, compounds that are cytotoxic or growth inhibitory, absorb light at 615 nm or precipitate.
Outcome: Compounds that showed >43% inhibition (>three standard deviations from the average compound inhibition) were defined as Active. Compounds that caused <=43% inhibition were defined as Inactive.
The following tiered scoring system has been implemented at SRMLSC. Compounds in this primary screen were scored on a scale of 0-40 based on activity where 100% inhibition corresponded to a score of 40 and no inhibition corresponded to a 0 score. A score of 18 or higher roughly correlates with the compound being active. In subsequent confirmatory dose response screens, active compounds are scored on a scale of 41-80 using an inverted linear correlation to EC50s. Compounds that do not confirm as actives in the dose response screen are given the score 0. In later stage probe development screening, active resynthesized confirmatory screen compounds and active analogues thereof score in a range of 81-100.
Data Table (Concise)