Screen for Chemicals that Shorten 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: 480
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. Compounds that reduce growth under restrictive conditions, but have little effect on growth under the control permissive condition are candidates for targeting lifespan regulatory pathways. An example of such a molecule is nicotinamide. 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), but does require concentrations in the millimolar range to have an effect in yeast.
From assays where cells grown under restrictive conditions were screened with compounds at 10 uM (AIDs 706 & 804), 759 compounds with significant growth-inhibitory effect were selected for confirmatory dose response assays under both restrictive and permissive conditions. The goal of the dose response assays was to identify compounds that selectively inhibit growth under restrictive conditions and therefore may be reducing the replicative lifespan of the cells. Here we describe the confirmatory dose response assay under restrictive conditions. The corresponding assay under permissive conditions is reported in a separate bioassay (AID 850). Compounds were screened in a 10-point 2-fold dilution series ranging from 0.098 to 50 uM. The percent inhibition of growth was calculated using the optical density in control wells with medium only (0% inhibition), and wells with cells treated with amphotericin B (100% inhibition). As a further control, cells treated with nicotinamide, which has a lifespan shortening effect on S. cerevisiae and hence has a selective growth inhibitory effect on cells grown under restrictive conditions, was included on each plate.
Preparation of assay
1. Cells (DeaD strain BB579) were streaked out on a YPGal agar plate and grown for 48 h at 30 degrees C.
2. 4 colonies were selected, 50 mL of YPGal medium in a flask was inoculated and grown at 30 degrees C 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 30 degrees C for 4 h. At the end of the pre-incubation, OD600 was measured for reference.
7. CSMM-D medium alone (negative control; 32 wells), amphotericin B (positive control; 24 wells), nicotinamide (control drug; 8 wells) and compounds in the presence of nicotinamide were plated with DMSO at 10 x concentration (final concentrations: amphotericin B 10 ug/mL, nicotinamide 1.5 mM, compounds 0.098-50 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 30 degrees C 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 121 degrees C 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
Data management: The percent inhibition of growth rate was calculated as:
100*(1-((Test Cmpd - Median Pos Ctrl)/(Median Neg Ctrl # Median Pos Ctrl)))
The upper limit of calculated inhibition was set to 100%.
From the % inhibition values of the different compound concentrations, the half maximal inhibitory concentration (IC50) was calculated using IDBS ActivityBase software and XLfit equation 205 for a four parameter logistic fit; the maximum and value was fixed at 100% while the minimum value was set to be to be <=0%. IC50s were extrapolated up to 100 uM.
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 >=30% inhibition for at least one concentration were defined as Active. If the inhibition at all doses was <30%, the compound was defined as Inactive.
The following tiered scoring system has been implemented at SRMLSC. Compounds in the primary screen were scored on a scale of 0-40. In this confirmatory dose response screen, active compounds were scored on a scale of 41-80 using an inverted logarithmic correlation to IC50s. (Activity score=41 + 13*(- log[IC50] + 2), where the IC50 is expressed in micromolar (uM) concentration and values of "<0.098" and ">100" were counted as 0.098 and 100 uM, respectively). Compounds that did not confirm as Actives in this 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)