Screen for Chemicals that Extend Yeast Lifespan, Dose Response
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: 245
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 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 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. A single dose screen of the MLSMR was performed (AID 775) from which 500 active compounds were selected for a dose response assay. In addition to the confirmatory screen described here, the same compounds were also tested in dose response screens for their ability to extend lifespan in the same strain in the absence of exogenously added nicotinamide (AID 812) and their ability to act as false positives through activation of the Gal1p promoter (AID 816).
Compounds were screened in an 8-point 2-fold dilution series ranging from 0.078 to 10 uM in the presence of 1.5 mM nicotinamide. The percent activation of lifespan, 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% activation), and wells with cells grown in medium without nicotinamide as an indicator of 100% activation. From the % activation values of the different compound concentrations, the half maximal effective concentration (EC50) was calculated using IDBS ActivityBase software and XLfit equation 205 for a four parameter logistic fit; the maximum and minimum values were fixed at 100% and 0%.
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. Nicotinamide (negative control), CSMM-D 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 0.078-10 uM, DMSO 0.5%) 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 absorb light at 615 nm.
Outcome: An activity threshold of EC50 <=10 uM was set and compounds that met this criterion are defined as Active. Compounds that exhibited an EC50 >10 uM are defined as Inactive.
The following tiered scoring system has been implemented at SRMLSC. Compounds in the primary screen (AID 775) were scored on a scale of 0-40 based on activity. In this confirmatory dose response screen active compounds were scored on a scale of 41-80 using an inverted linear correlation to EC50s between 0 and 10 uM. Compounds that did not confirm as actives in the dose response screen were given the score 0. In later stage probe development screening, active resynthesized confirmatory screen compounds and active analogues thereof will score in a range of 81-100.
Data Table (Concise)