uHTS Fluorescent assay for identification of inhibitors of Apaf-1
Apoptosis is a major form of programmed cell death that multicellular organisms utilize to maintain tissue homeostasis and to eliminate unwanted or damaged cells. It plays a critical role in development, immune responses and many other physiological events. In mammals, the mitochondrial cytochrome c-mediated apoptotic pathway is initiated by cytochrome c release from mitochondria. Deregulation of the cytochrome c apoptotic pathway can lead to diseases such as cancer, immune disorders, and neurodegenerative diseases. ..more
BioActive Compounds: 2353
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBIMR, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: R01 CA136513
Assay Provider: Dr. Xuejun Jiang, Sloan-Kettering Institute for Cancer Research, New York, NY
Apoptosis is a major form of programmed cell death that multicellular organisms utilize to maintain tissue homeostasis and to eliminate unwanted or damaged cells. It plays a critical role in development, immune responses and many other physiological events. In mammals, the mitochondrial cytochrome c-mediated apoptotic pathway is initiated by cytochrome c release from mitochondria. Deregulation of the cytochrome c apoptotic pathway can lead to diseases such as cancer, immune disorders, and neurodegenerative diseases.
The overall goal of this project is to identify Apaf-1 inhibitors by high throughput screening. Apaf-1 is the essential mediator of the mitochondrial cytochrome c-mediated caspase activation pathway. In this pathway, various apoptotic stimuli can cause cytochrome c release from mitochondria. The released cytochrome c binds to and activates Apaf-1 to trigger formation of the multiprotein complex, the apoptosome. Apoptosome complex subsequently induces activation of caspases, the executioners of apoptosis. Therefore inhibition of Apaf-1 function can block caspase activation in the upstream of the pathway.
In this biochemical fluorescent assay, the Apaf-dependent activation of caspase-9 and consequently caspase-3 is detected based on ability of the activated caspase-3 to cleave a fluorogenic rhodamine 110 -labeled DEVD tetra-peptide, generating a strongly fluorescent Rh110 that is monitored fluorimetrically.
1. Jiang X, Wang X. Cytochrome c Promotes Caspase-9 Activation by Inducing Nucleotide Binding to
Apaf-1. J. Biol. Chem. 2000 (275): 31199-31203.
2. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. Three-Dimensional Structure of the
Apoptosome: Implications for Assembly, Procaspase-9 Binding, and Activation. Molecular Cell 2002
3. Jiang X, Kim H, Shu H, Zhao Y, Zhang H, Kofron J, Donnelly J, Burns D, Ng S, Rosenberg S, Wang
X. Distinctive Roles of PHAP Proteins and Prothymosin-a in a Death Regulatory Pathway. Science
2003 (299): 223-226.
4. Yin Q, Park HH, Chung JY, Lin SC, Lo YC, da Graca LS, Jiang X, Wu H. Caspase-9 holoenzyme is a
specific and optimal pro-caspase-3 processing machine. Molecular Cell 2006 (22) 259-268.
5. Gao Z, Tian Y, Wang J, Yin Q, Wu H, Li Y, Jiang X. A Dimeric Smac/Diablo Peptide Directly Relieves
Caspase-3 Inhibition by XIAP: Dynamic and Cooperative Regulation of XIAP by Smac/Diablo. J. Biol.
Chem. 2007 (282) 30718-30727
1) Apaf-1, cytochrome c, procaspase-9, and procaspase-3 proteins were obtained from Dr. Jiang's laboratory.
2) Assay Buffer: 20 mM Hepes pH 7.5, 15 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 20 mM B-Mercaptoethanol, 5% Sucrose, 0.1% CHAPS, 0.1% BSA
3) (Z - DEVD)2 - Rh110 tetrapeptide substrate was purchased from Anaspec (Catalog#: 60304-5); DEVD-CHO was purchased from Calbiochem (Cat# 235420)
4) Corning 1536 well black solid bottom plate (Cat# 3724)
1) Using LabCyte Echo, pre-dispense 40 nL of test compounds from a 2 mM compound source plate into assay plate Col. 5-48, and 40 nL of 0.5 mM DEVD-CHO in DMSO into col. 1-2, and 40 nL of DMSO into Col. 3-4
2) Using Thermo Scientific MultiDrop Combi nL, dispense 1.5 uL of 2.7 nM of Apaf-1 in Assay Buffer into all columns 1-48.
3) Using Thermo Scientific MultiDrop Combi nL, dispense 1.5 uL of Mix 2, containing 200 uM dATP and 53.3 nM Procaspase-3 in Assay Buffer, into all columns 1-48.
4) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
5) Incubate for 30 minutes at room temp.
6) Using Thermo Scientific MultiDrop Combi nL, dispense 1 uL of Mix 3, containing 0.67 uM Cytochrome C, 53.3 nM Caspase-9, and 120 uM DEVD-Rho in Assay Buffer, into columns 1-48.
7) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
8) Incubate for 50 minutes at room temp.
9) Read plates on Perkin Elmer Viewlux in Fluorescence mode (Ex: 480 nm; Em: 540 nm).
10) Data analysis was performed using CBIS software (ChemInnovations, Inc).
Compounds that demonstrated an activation of >= 40% at 20 uM concentration 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.
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 screening data. The score is correlated with % activity in the assay:
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
Scoring for Single concentration confirmation screening is not applicable to this assay.
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)