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BioAssay: AID 812

Screen for Chemicals that Extend Yeast Lifespan; Lifespan Extension in the Absence of Nicotinamide Secondary Screen

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 ..
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 Tested Compounds
 Tested Compounds
All(499)
 
 
Active(3)
 
 
Inactive(494)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(499)
 
 
Active(3)
 
 
Inactive(494)
 
 
Inconclusive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 812
Data Source: SRMLSC (YDOD LS Ext DR NoNam BB579)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-10-01
Modify Date: 2007-11-16

Data Table ( Complete ):           Active    All
BioActive Compounds: 3
Depositor Specified Assays
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AIDNameTypeComment
705Yeast Lifespan Shortening Chemical Screening, Permissive Growth Control - Pilot Screenscreening
706Yeast Lifespan Shortening Chemical Screening, Restrictive Growth Control - Pilot Screenscreening
775Screen for Chemicals that Extend Yeast Lifespanscreening
804Screen for Chemicals that Shorten Yeast Lifespanscreening
809Screen for Chemicals that Extend Yeast Lifespan, Dose Responseconfirmatory
816Screen for Chemicals that Extend Yeast Lifespan; Counterscreen for Compounds that Activate the Gal1p promoterconfirmatory
849Screen for Chemicals that Shorten Yeast Lifespan, Dose responseconfirmatory
850Screen for Chemicals that Shorten Yeast Lifespan, Dose Response Permissive Growth Controlconfirmatory
434994A yeast HTS for caloric restriction mimetics that inhibit age-related superoxide tested on cherry picked compoundsscreening
449753Confirmation dose response of hits from high-throughput yeast screen for caloric restriction mimetics that inhibit age-related superoxideconfirmatory
435000Counterscreen for compounds affecting dihydroethidium (DHE) oxidation and fluorescencescreening
489042Phenotypic dose response assay for compound effect on Saccharomyes cerevisiae proliferationother
2690A yeast HTS for caloric restriction mimetics that inhibit age-related superoxidescreening
2706A yeast HTS for caloric restriction mimetics that inhibit age-related superoxide for Validation Compound Setscreening
Description:
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 a confirmatory dose response assay (AID 809). In the secondary screen described here, the same 500 compounds were tested in a dose response screen for their ability to extend lifespan in the DeaD strain in the absence of exogenously added nicotinamide.

Compounds were screened in a 10-point 2-fold dilution series ranging from 0.078 to 40 uM in restrictive CSMM-D 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. While galactose does not extend lifespan it allows the cells to overcome the lifespan restrictive growth by activation of the Gal1p::Cdc6 locus. 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%.
Protocol
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 0.0005 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. 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 42 h incubation, plates were shaken for 30 s and OD615 was read in an EnVision (PerkinElmer) multilabel plate reader.

Media Prep

YPGal medium
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
Filter sterilize
Comment
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. Compounds that exhibited an EC50 >40 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 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 ModifierString
2EC50FloatμM
3EC50 Hill SlopeFloat
4EC50 Std Dev ModifierString
5EC50 Std DevFloat
6EC50 NormChi2Float
7Max ActivationMaximum activation at any concentrationFloat%
8Max Activation ConcConcentration at which maximum activation was observed.FloatμM
9Min ActivationMinimum activation at any concentrationFloat%
10Min Activation ConcConcentration at which minimum activation was observed.FloatμM
11Activation @ 40 uMFloat%
12Activation @ 20 uMFloat%
13Activation @ 10 uMFloat%
14Activation @ 5 uMFloat%
15Activation @ 2.5 uMFloat%
16Activation @ 1.25 uMFloat%
17Activation @ 0.625 uMFloat%
18Activation @ 0.313 uMFloat%
19Activation @ 0.156uMFloat%
20Activation @ 0.078 uMFloat%

Data Table (Concise)
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