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BioAssay: AID 868

Screen for Chemicals that Inhibit the RAM Network

The RAM network is a novel and highly conserved signaling pathway involved in maintenance of polarized growth, cell proliferation, and control of gene expression. The pathway is well conserved between yeast and metazoans. In budding yeast, where the pathway was discovered and is best characterized, it is absolutely required for transcription of genes whose expression is driven by the transcription factor Ace2 (Weiss et al., 2002; Nelson et al., 2003). ..more
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 Tested Compounds
 Tested Compounds
All(194553)
 
 
Active(3545)
 
 
Inactive(191008)
 
 
 Tested Substances
 Tested Substances
All(194582)
 
 
Active(3545)
 
 
Inactive(191037)
 
 
 Related BioAssays
 Related BioAssays
AID: 868
Data Source: SRMLSC (Yeast RAM SD FS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-04
Modify Date: 2008-06-03

Data Table ( Complete ):           Active    All
BioActive Compounds: 3545
Depositor Specified Assays
AIDNameTypeComment
1298A screen for inhibitors of the budding yeast RAM network using a sensitized drug exporter inhibited strainscreening
1305Screen for Chemicals that Inhibit the RAM Network - Dose Response with Drug Efflux Deficient Sensitized Control Strainconfirmatory
1307Screen for Chemicals that Inhibit the RAM Network - Confirmatory Dose Responseconfirmatory
869Screen for Chemicals that Inhibit the RAM Network, Pilot Screenscreening
Description:
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Eric Weiss, Northwestern University
Award: 1R21NS056968-01

A screen for inhibitors of the budding yeast RAM network

The RAM network is a novel and highly conserved signaling pathway involved in maintenance of polarized growth, cell proliferation, and control of gene expression. The pathway is well conserved between yeast and metazoans. In budding yeast, where the pathway was discovered and is best characterized, it is absolutely required for transcription of genes whose expression is driven by the transcription factor Ace2 (Weiss et al., 2002; Nelson et al., 2003).

A genetic selection for loss of RAM network function was created by placing the URA3 gene under the control of the Ace2-driven DSE1 promoter. In this strain, URA3 expression depends on RAM network function. The URA3 gene encodes an enzyme required for uracil biosynthesis. Importantly, this enzyme also converts the innocuous compound 5-fluoroorotic acid (5-FOA) to 5-fluorouracil, which is highly toxic. Hence, 5-FOA kills these cells unless RAM network function is blocked.

This dependency was previously exploited in an exceptionally powerful conventional genetic selection to isolate hundreds of new loss-of-function alleles of RAM network components. Approximately 600,000 colonies were screened in a single step, resulting in ~500 primary isolates. Phenotype-based secondary screening was straightforward: cells lacking RAM network function grow as fast-sinking clumps. Of ~500 primary isolates, ~400 were new RAM network alleles. Thus, the screening approach is remarkably selective.

Here, this system was used for selection-based screening of compound libraries to discover highly specific small molecule inhibitors of the RAM network. The yeast strain used in the screen was engineered to contain an "analog-sensitive" mutant allele (Bishop et al.) of the RAM network protein kinase Cbk1 (Cbk1-as), which is susceptible to inhibition by 1-napthyl PP1 (1-NA-PP1)(Weiss et al., 2002). This permitted pre-inhibition of URA3 expression, a step that was necessary to ensure that cells did not begin growth under screening conditions with lethal levels of this gene product, and also allowed the use of 1-NA-PP1 as a positive control drug.

Percent inhibition of the RAM network signaling was calculated using the optical density in control wells with untreated cells as full RAM network activity (no growth; 0% inhibition) and wells treated with 10 uM 1-NA-PP1 as complete inhibition of the RAM network (full growth; 100% inhibition). Library compounds were screened at 50 uM.
Protocol
Protocol:
1. Inoculate cells (homozygous diploid MATa/MATalpha cbk1-as2/cbk1-as2 dse1pr::URA3/dse1pr::URA3) from glycerol stock into synthetic complete medium without uracil (10 mL volume), grow in shaker at 30 degrees C for 6-8 h.
2. Dilute to OD 0.001 in synthetic complete medium with uracil and 25 uM 1-NA-PP1 (25 mL final volume). Grow in shaker at 30 degrees C over night.
3. The following day, plate 5 uL of 10x concentrated compounds (500 uM in 5% DMSO; final concentration 50 uM in 0.5% DMSO), positive control (1-NA-PP1: 100 uM in 5% DMSO; final concentration 10 uM in 0.5% DMSO) and negative control (5% DMSO; final concentration 0.5%) in the wells of 384-well plates.
4. Read OD600 of the yeast culture. The OD should be below 1.0 to ensure that the cells are in log phase. Dilute to OD600 0.002 in synthetic complete medium with 1 mg/mL 5-FOA, and plate into the 384-well plates (45 uL/well).
5. Grow at 30 degrees C in humidified chamber for 2 days.
6. Read OD615 on PerkinElmer EnVision multilabel plate reader.


Growth Media

Synthetic complete medium:

6.7 g yeast nitrogen base (Sigma Y0626)
- Dissolve in 800 mL water and autoclave.
- Let cool down to <37 degrees C and add:
2.0 g complete drop-out mix (USBio D9515)
10 mL adenine supplement (8 mg/mL)
100 mL 20% (w/v) dextrose
Water to 1.0 L, make sure that components of drop-out mix are in solution
- Filter sterilize (0.2 um)


Synthetic complete medium without uracil:

6.7 g yeast nitrogen base (Sigma Y0626)
- Dissolve in 800 mL water and autoclave.
- Let cool down to <37 degrees C and add:
2.0 g drop-out mix without uracil (USBio D9535)
10 mL adenine supplement (8 mg/mL)
100 mL 20% (w/v) glucose
Water to 1.0 L, make sure that components of drop-out mix are in solution
- Filter sterilize (0.2 um)


Synthetic complete medium with 5-FOA:

6.7 g yeast nitrogen base (Sigma Y0626)
1.0 g 5-FOA (US Biological F5050)
- Dissolve in 800 mL water and autoclave.
- Let cool down to <37 degrees C and add:
2.0 g complete drop-out mix (USBio D9515)
10 mL adenine supplement (8 mg/mL)
100 mL 20% (w/v) glucose
Water to 1.0 L, make sure that components of drop-out mix are in solution
- Filter sterilize (0.2 um)


1-NA-PP1 stock, 25 mM (1,000-2,500x)

- 1-NA-PP1 from Toronto Research Chemicals (A603004)
- Dissolve 10 mg in 1.26 mL DMSO. Aliquot and store in desiccator at -20 degrees C


Data management:
Percent inhibition was calculated as:

100*([ODcompound-ODneg.ctrl.]/[ODpos.ctrl.-ODneg.ctrl.])
Comment
Comment:
Possible artifacts in this assay include, but are not limited to, compounds that inhibit either the Ura3 protein or the mutant Cbk1-as allele specifically (and not a wild type Cbk1 protein), that block uptake of 5-FOA or promote its efflux, or that absorb light at 615 nm or precipitate.

Outcome: Compounds that showed >8.4% inhibition (>three standard deviations from the average compound inhibition; average inhibition: 1.71%, one standard deviation: 2.24%) were defined as Active. Compounds that caused <=8.4% 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 the lowest activity from the two runs where 0 corresponds to no inhibitory activity and a score of 40 corresponds to 100% inhibition. An activity score of 4 or higher corresponds to compounds with an active outcome. In a subsequent confirmatory dose response screen, active compounds will be scored on a scale of 41-80 while compounds that do not confirm as actives will be 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
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (50μM**)Float%
2Inhibition (replicate) (50μM**)Float%

** Test Concentration.

Data Table (Concise)
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