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BioAssay: AID 1362

Chemical Genetic Screen to Identify Inhibitors of Mitochondrial Fusion - Primary Screen

Screening for compounds that inhibit mitochondrial fusion using a yeast model system as a primary screening tool ..more
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AID: 1362
Data Source: SRMLSC (YMitoFus_SD)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-07-14
Modify Date: 2008-07-17

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 4011
Related Experiments
1361Chemical Genetic Screen to Identify Inhibitors of Mitochondrial Fusion - Confirmatory ScreenConfirmatorydepositor-specified cross reference
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Jodi M. Nunnari, University of California, Davis
Award: R03 MH081279-01

Screening for compounds that inhibit mitochondrial fusion using a yeast model system as a primary screening tool

Mitochondria are essential, double-membraned organelles that perform a myriad of tasks within cells. Unlike their bacterial ancestors, they are not discrete entities; rather isolated organelles are transient and are constantly in communication via fusion to form both localized and widespread mitochondrial syncytia within cells. Mitochondrial fission antagonizes fusion and together these events function to create a compartment that is connected and thus functional, yet able to be distributed to distant cellular destinations via transport on actin or microtubule networks. Using a genetic approach in S. cerevisiae, we have identified proteins that are required for mitochondrial fission and fusion (Hoppins et al, 2007).

Remarkably, the key proteins among these are three dynamin-related proteins (DRPs), which are large self-assembling GTPases that regulate membrane dynamics in a variety of cellular processes. Orthologs of these DRPs exist in mammalian cells, indicating the remarkable conservation of these processes. Indeed, our work in yeast has helped to form the foundation for studies showing that the human mitochondrial fission and fusion orthologs play important and intimate roles in apoptosis (Hoppins et al, 2007). Mutations in the human mitochondrial fusion DRPs result in the neurodegenerative diseases, Charcot-Marie Tooth and dominant optic atrophy, underscoring the physiological importance of mitochondrial dynamics in humans (Hoppins et al, 2007).

Here we use chemical genetics to gain insight into the mechanisms and physiological roles of mitochondrial dynamics. We expect to identify small molecules that inhibit mitochondrial fusion by screening the MLSMR chemical library using a straightforward growth-based assay in S. cerevisiae strains engineered so that growth reports on mitochondrial fusion activity. Specifically, we are searching for molecules that inhibit the growth of a wild-type strain but not a fission mutant strain (delta-dnm1) in a non-fermentable growth medium (Hoppins et al, 2007).

The approach has proven successful in a smaller screen for inhibitors of mitochondrial division. From that screen, a small molecule was identified that specifically inhibits the DRP Dnm1 and thereby inhibited mitochondrial division in both yeast and mammalian cells (Cassidy-Stone et al, 2008).
Preparation of assay
- "Wild type" JNY 913 (ade2-1; leu2-3;; trp1-1; ura3-1; can1-100 pdr1::KANr, pdr3::HIS5) and "mutant" JNY 915 (ade2-1; leu2-3;; trp1-; ura3-1; can1-100 pdr1::KANr, pdr3::HIS5, dnm1::HIS3) cells were streaked out from frozen stock onto YPEG agar plates.
- The plates were grown inverted for 3 days at 30 degrees C. Plates were then stored (sealed with parafilm) and used up to 2 weeks at 4 degrees C before being replaced with a fresh plate.
- 4 colonies from each strain were selected and inoculated in 10 mL of YPEG medium in a flask, and grown 6-8 h at 30 degrees C with shaking at 260 rpm (Primary Culture).
- 25 mL of YPEG was inoculated with primary culture to OD600 of 0.04 and grown for 16-20 h at 30 degrees C with shaking at 260 rpm (Secondary Culture).
- The culture was confirmed to be in mid to late log phase. To qualify, the OD600 needed to be below 2; ideally between 0.5 and 0.9.
- Secondary culture was diluted to OD600 of 0.007 in YPEG medium and 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.
- YPEG medium alone (negative control), amphotericin B (positive control), 3-[6-(ter-butyl)-1,1-dimethyl-2,3-dihydro-1H-inden-4-yl]-5-(trifluoromethyl)-1H-pyrazole (reference control drug that has a selective effect on the wild type strain) and compounds were plated with DMSO at 6 x concentration (final concentrations: amphotericin B 10 ug/mL, control drug 20 uM, compounds 10 uM, DMSO 0.1%) in duplicate 384-well plates: 5 uL/well.
- The two yeast strains were added to each of the two plate sets: 25 uL/well. Plates were incubated at 30 degrees C in a humidified chamber.
- After 24 h incubation, 30 uL of CellTiter-Glo (Promega) was added to the cells and the plates incubated at room temperature for 1 h. Luminescence was read in an EnVision (PerkinElmer) multilabel plate reader.

Growth Media

YPEG agar plates (1.0 L):
20 g peptone (Difco DF0118-17-0)
10 g yeast extract (Difco DF0127-17-9)
20 g agar
Milli-Q water up to 855 mL
Autoclave at 121 degrees C for 20 min
5 mL sterile 8 mg/mL tryptophan
10 mL sterile 6 mg/mL adenine sulfate
30 mL 100% ethanol
100 mL sterile 30% glycerol

YPEG medium (1.0 L):
20 g peptone (Difco DF0118-17-0)
10 g yeast extract (Difco DF0127-17-9)
Milli-Q water up to 855 mL
Autoclave at 121 degrees C for 20 min
5 mL sterile 8 mg/mL tryptophan
10 mL sterile 6 mg/mL adenine sulfate
30 mL 100% ethanol
100 mL sterile 30% glycerol

Data management:
The percent inhibition of growth 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%.
Possible artifacts in this assay include, but are not limited to, compounds that are cytotoxic, interfere with the luminescence reaction, absorb luminescence or precipitate.

Outcome: Compounds that showed >57.94% inhibition (>three standard deviations from the average compound inhibition; average inhibition: 3.79%, one standard deviation: 17.88%) in the wild type strain AND had higher activity in the wild type strain than in the mutatnt stran were defined as Active. Compounds that caused <=57.94% inhibition were defined as Inactive. In the case when compounds were screened more than once, the outcome was calculated based on the average inhibition

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 inactive compounds and a score of 40 corresponds to the most selective and potent compound. The score was calculated as follows:

Score = 0.5+39.5*Log10[(1+inhib(wt)-inhib(mut))*inhib(wt)/200.5]

For this calculation, the inhibition values were limited to be between 0 and 100.

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
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1WT_Inhibition @ 10 uM Avg (10μM**)Float%
2WT_Inhibition @ 10 uM Rep 1 (10μM**)Float%
3WT_Inhibition @ 10 uM Rep 2 (10μM**)Float%
4WT_Inhibition @ 10 uM Rep 3 (10μM**)Float%
5Mut_Inhibition @ 10 uM Avg (10μM**)Float%
6Mut_Inhibition @ 10 uM Rep 1 (10μM**)Float%
7Mut_Inhibition @ 10 uM Rep 2 (10μM**)Float%
8Mut_Inhibition @ 10 uM Rep 3 (10μM**)Float%
9VerificationData has been verifiedString

** Test Concentration.

Data Table (Concise)
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