Single concentration confirmation of small molecule inhibitors of tim10-1 yeast via a luminescent assay
Defects in mitochondrial assembly impact a wide range of diseases from degenerative muscle and neural diseases to cancer (Wallace, 2005). The mitochondrion is not only important for the production of energy but plays an important role in other aspects such as intermediary metabolism and signaling. The mitochondrion contains an inner membrane and outer membrane that separate the matrix from the more ..
BioActive Compounds: 4139
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 DA027714-01A1
Assay Provider: Dr. Carla Koehler, University of California, Los Angeles, CA (UCLA)
Defects in mitochondrial assembly impact a wide range of diseases from degenerative muscle and neural diseases to cancer (Wallace, 2005). The mitochondrion is not only important for the production of energy but plays an important role in other aspects such as intermediary metabolism and signaling. The mitochondrion contains an inner membrane and outer membrane that separate the matrix from the intermembrane space. Proteins destined for the mitochondrion are imported via the Translocase of the Outer Membrane (TOM) and the Translocases of the Inner Membrane (TIM23 for proteins destined for the matrix and TIM22 for proteins destined for the inner membrane) (Koehler, 2004). This HTS campaign focuses on the characterization of small molecule inhibitors that modulate the TIM22 import pathway, using yeast as a model. Components in this pathway consist of soluble components in the intermembrane space and an insertion complex in the inner membrane.
The goal of this screen is to discover small molecule modulators that affect the TIM22 import pathway by taking advantage of a yeast temperature sensitive (ts) mutant tim10-1 (Koehler et al, 1998) Specifically, the yeast ts tim10-1 mutant grows at the permissive temperature of 25 oC but ceases growth at 37 oC. We have developed a growth assay based on identifying compounds that cause the tim10-1 mutant to arrest growth at 25 oC. Thus, we will identify small molecules that were synthetic lethal with the tim10-1 mutant at the permissive temperature, rationalizing that these probes would target the already compromised TIM22 import pathway.
This goal of this assay is to confirm hits from "uHTS identification of small molecule inhibitors of tim10-1 yeast via a luminescent-based reporter assay", AID 463190.
Wallace DC (2005) A Mitochondrial Paradigm of Metabolic and Degenerative Diseases, Aging, and Cancer: A Dawn for Evolutionary Medicine. Annu Rev Genet.
Koehler CM (2004) New developments in mitochondrial assembly. Ann. Rev. Cell Dev. Biol., 20, 309-335.
Koehler CM, Jarosch E, Tokatlidis K, Schmid K, Schweyen RJ, Schatz G (1998) Import of mitochondrial carriers mediated by essential proteins of the intermembrane space. Science, 279, 369-373.
Yeast Strain: ySHDSTim10ts
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)
I. Compound Addition:
1. Using LabCyte Echo, transfer 40 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.
II. Set up of tim10-1 assay:
The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).
3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 2000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25 ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells.
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.
IV. Reading plates:
12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.
Selected compounds from AID 463190 were retested in duplicate at 20 uM. Compounds with %Activity >= 32% for either replicate are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)