Screen for Chemicals that Extend Yeast Lifespan; Counterscreen for Compounds that Activate the Gal1p promoter
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 ..
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 (BB579) 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 499 active compounds were selected for primary screen confirmatory (AID 809) as well as a secondary (without nicotinamide; AID 812) dose response assay. In the DeaD assay, one potential cause of false positives is activation of the Gal1p::Cdc6 locus, which would allow the cells to grow in a non-lifespan restricted fashion. To address this, we performed a screen with a strain, BB569, which has the Gal1p::Cdc6 locus but lacks the HO::Cdc6 locus that allows for lifespan restricted growth in the absence of galactose in the DeaD strain. Hence, in restrictive medium, the BB569 strain will not grow at all and can be used to screen for compounds that activate the Gal1p promoter.
Compounds were screened in a 10-point 2-fold dilution series ranging from 0.078 to 40 uM in restrictive CSMM-RD medium. The percent activation of lifespan was calculated using the optical density in control wells with cells in restrictive medium as 0% activation and wells with cells grown in restrictive medium with 2.5% galactose 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 (Gal1p::Cdc6 strain BB569) 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-RD restrictive growth medium. OD600 was measured again. The culture was diluted to an OD600 of 0.002 in CSMM-RD 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-RD restrictive medium alone (negative control), medium with galactose (positive control) and compounds were plated with DMSO at 10 x concentration (final concentrations: galactose 2.5%, compounds 0.078-40 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 24 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-RD (Complete Synthetic Minimal Medium-Raffinose/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) raffinose
25 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 <=40 uM was set and compounds that met this criterion are defined as Active. In this counterscreen, since none of the tested compounds exhibited an EC50 <=40 uM, all of them were Inactive. This means that none of the Actives in the primary and secondary screen dose response assays (see Related BioAssay Links) were so because of activation of the Gal1p promoter and likely not because of compound precipitation or light absorption either.
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 dose response counterscreen active compounds were scored on a scale of 41-80 using an inverted linear correlation to EC50s between 0 and 40 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)