Screen for compounds that selectively inhibit cytochrome c release from purified mitochondria (CytoCRel)
Cytochrome c release from the mitochondria into the cytoplasm is a potent physiologic stimulus for caspase-9 and caspase-3 activation. Cytochrome c release is a shared feature of many neurologic disorders, including acute forms such as stroke as well as chronic forms such as amyotrophic lateral sclerosis. Many factors could lead to cytochrome c release from mitochondria, such as Ca2+, reactive more ..
Cytochrome c release from the mitochondria into the cytoplasm is a potent physiologic stimulus for caspase-9 and caspase-3 activation. Cytochrome c release is a shared feature of many neurologic disorders, including acute forms such as stroke as well as chronic forms such as amyotrophic lateral sclerosis. Many factors could lead to cytochrome c release from mitochondria, such as Ca2+, reactive oxygen species and Bid/tBid protein in vitro and in vivo. We have developed an in vitro assay of cytochrome c release using purified mitochondria induced by Ca2+ and/or Bid/tBid protein. The quantity of released cytochrome c is measured by ELISA in 96 well plate. Compounds that decrease the release of cytochrome c under apoptotic challenge in vitro maybe also behave the same way in vivo and thus protect neuronal cell from dying.
Screen: An aliquot of 100 uL of (0.1 mg/ml) mouse liver mitochondrial preparation was preincubated with compounds from the NINDS library at a final concentration of 20 uM (diluted with assay medium by 500x from the 10 mM stock solution in DMSO) for 5 min in MRM buffer. Mitochondria were then incubated with 100 uM CaCl2 at 30oC for 30 min (CaCl2). Mixtures were centrifuged at 10,000g at 4oC for 10 min and the supernatant evaluated by Cytochrome c ELISA kit.
Cell Lines: C57B6 mice
Assay Medium: MRM buffer: 250 mM sucrose, 10 mM Hepes, pH7.5, 1 mM ATP, 5 mM sodium succinate, 80 uM ADP, 2 mM K2HPO4
Assay Reagent: Cytochrome c ELISA kit:RandD System MCTC0
Data Analysis: Each of 13 drug kits was evalated with a separate mitochondrial preparation on a separate 96-well elisa plate. OD value was obtained for each sample by subtracting background 540 nm absorbance reading from 450 nm absorbance reading. The OD values were converted to cytochrome c concentration according to the 6-point standard curve obtained from each plate. Distribution curves of cytochrome c release were obtained for each plate. Normal distributions were seen with peak supposedly showing the majority of drugs without any effect on cytochrome c release, with drugs showing protection to the left and drugs showing toxicity to the right of the curve peak. Average values of cytochrome c release of all samples from each plate was calculated for each plate. The values over 2 times the plate average were dropped as toxic and new-average was calculated for each plate. The values from each plate were normalized according to plate new-average considered as 100% of cytochrome c release. The normalized values are presented as the final output.
Dilution Series: 48 compounds were selected for retesting in 10 micro-M and 20 micro-M to choose best 12 drugs. The assay was performed as described in the screening section. We have also tested selected 12 drugs effect in Cacium- and Bid-induced cytochrome c release in 1 micro-M and 10 micro-M concentration.
Assay Type in vitro cytochrome c release from mitochondria
Number Replicates 1
Assay Readout Absorbance at 450nm substract Absorbance at 540nm
Detection Instrument Labsystems Multiskan MCC/340 platereader
Concentration Primary Screen (uM) 20
Number Negative Controls 0
Negative Control Mean 0.0
Negative Control Standard Deviation 0.0
Number Positive Controls 0
Positive Control Mean 0.0
Positive Control Standard Deviation 0.0
A Note about the Activity Matrix Rankings:
The data for each assay has been converted into a numerical ranking of all 1040 compounds for that assay. Thus, a compound with a rank of 1 was the top scoring compound in that assay and a compound with a rank of 1040 was the lowest scoring compound in that assay. Compounds with equal scoring were given equal rank.
PubChem scores were derived as (1040-NINDS activity rank). Substances with reported EC50 values were considered active.
PI Name Robert M Friedlander
Affiliation Brigham and Womens Hospital
Investigator Martin Drozda
Investigator Shan Zhu
Data Table (Concise)