Assay to identify inhibitors of polyglutamine-induced caspase-3 activation (CASP3)
This Assay identifies inhibitors of polyglutamine-induced caspase-3 activation. Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked recessive neurodegenerative disorder due to an expansion of the CAG triplet repeat sequence within exon 1 of the androgen receptor gene. Evidence indicates that expression of the androgen receptor with an expanded polyglutamine stretch induces apoptosis in more ..
This Assay identifies inhibitors of polyglutamine-induced caspase-3 activation. Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked recessive neurodegenerative disorder due to an expansion of the CAG triplet repeat sequence within exon 1 of the androgen receptor gene. Evidence indicates that expression of the androgen receptor with an expanded polyglutamine stretch induces apoptosis in culture cells.The androgen receptor is cleaved by caspase-3 and it has been proposed that caspase cleavage plays a central role in the induction of neural cell death in SBMA. Inhibition of caspase activation is an attractive strategy for the therapy of many neurological diseases. Since caspase-3 is one of the most reproducible and reliable marker of toxicity, we examined the Caspase-3 activity of HEK 293 cells transiently expressing expanded form of AR to find compounds that inhibit poly-Q induced Caspase-3 activation.
Screening - incubation of cells with test compounds. For the caspase-3 assay, cells were seeded in 48-well assay plates (Costar, #3548) 1 ml/well for a cell density of 120,000 cells/well the day prior to transfection. Transfection with 0.2 micro-g of DNA was carried out using Lipofectamine Plus following the manufacturer's protocol (Gibco BRL).After transfection cells are incubated for 16 hours. Compounds from the NINDS library were added to reach a final concentration of 10 uM (from the 2 mM stock solution in DMSO). The cells were incubated with the test compounds at 37 degrees C for ~48 hours. After incubation, the medium and compounds were removed from the wells and the cells were resuspended in 100 uL of lysis Buffer. The cell lysates were stored at -80 degrees C o/n and then they were kept in the dark for 2 hours at 37 degrees C with 50 uM of fluorogenic substrate Ac-DEVD-AFC (Biosource) in a total volume of 200 uL of assay buffer. Substrate cleavage was detected using Cytofluor II Fluorescence multiwell plate reader (PerSeptive Biosystems) with excitation and emission wavelengths of 420 and 520 nm, respectively. The specificity of the Caspase-3 activity has been confirmed by inhibition with Z-VAD-FMK (PharMingen) at 20 uM. used as positive control.
Cell Lines: HEK-293 human embryonic kidney cells were transiently transfected with cDNA encoding the truncated form of the androgen receptor with 112 CAG repeats.
Growth Medium: HEK-293 cells were maintained in DMEM medium (Gibco BRL) supplemented with 10% (v/v) Fetal Bovine Serum, 100U/ml penicillin/100 micro-g/ml streptomycin and 2 mM L-glutamine.
Assay Medium: The cell culture medium used for the caspase-3 assay was identical to the growth medium.
Assay Reagent: Caspase-3 assay buffers: LYSIS BUFFER: 10 mM TRIS, pH 7.3, 10 mM NaH2PO4, 150 mM NaCl, 1% Triton X-1002x ASSAY BUFFER 20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 20% glycerol.
Data Analysis: The caspase-3 assay was subjected to validation to assess inter-well and intra-plate variability and reproducibility of the assay.Each compound library plate was tested once.The signal from each compound-treated well was compared to the average of 3 control wells on the same 48-well plate. The drugs exhibiting a caspase-3 inhibition greater than 1 standard deviation from the mean were retested in triplicate in two separate experiments.
Dilution Series: 10 compounds were selected for retesting in a 5-point dilution series over a range of 10 nM to 50 micro-M. Once set up, the assay was performed in triplicate and repeated in three separate experiments. Concentrations required to achieve 50% of the maximally achievable effect for each compound (EC50) were calculated using GraphPad Prism.
Detection Reagent: Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin Ac-DEVD-AFC (Biosource #77-935)
Assay Type mammalian cell-based
Number Replicates 6
Assay Readout fluorescence
Detection Instrument Cytofluor II Fluorescence multiwell plate reader (PerSeptive Biosystems)
Liquid Handling Robot Not Applicable
Concentration Primary Screen (#M) 10
Number Negative Controls 78
Negative Control Mean 1002.0
Negative Control Standard Deviation 287.44
Number Positive Controls 78
Positive Control Mean 412.34
Positive Control Standard Deviation 124.21
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 Kenneth Fischbeck
Affiliation Neurogenetics Branch/NINDS/NIH
Investigator Federica Piccioni
Investigator J.Paul Taylor
Categorized Comment - additional comments and annotations
Data Table (Concise)