FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay
Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays. ..more
BioActive Compounds: 31
NCGC Assay Overview:
Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays.
Previous profiling work to determine the prevalence of compounds that affect FLuc enzymatic activity (PubChem AID 411) identified that ~3% of the MLSMR library (then at ~72K) inhibited FLuc (Auld et al., 2008). The MLSMR library was again profiled at a later date (when the library contained significantly more compounds - ~350K) for FLuc activity in a biochemical assay using purified FLuc in the presence of KM concentrations of substrates (D-LH2 and ATP), in an effort to identify compounds in the library that may act as competitive inhibitors. Of the compounds identified in this profile that inhibited FLuc, 151 compounds were selected for further analysis, in an effort to better understand their mode of inhibiting FLuc. The assay described here was to determine the activity of the compounds in a cell-based firefly luciferase reporter gene assay originally designed to identify compounds that enchanced translational read-through of a FLuc cDNA that contained a premature stop codon (Auld et al., 2009; Auld et al., 2010). Compounds that enhance translational read-through or compounds that stabilize FLuc intracellularly are identified as active compounds in the assay.
NCGC Assay Protocol Summary:
Reagents: Transient transfection of FlucUGA construct: TransFast Transfection Reagent (Promega #E2431); FLucUGA construct (refer to Auld et al., 2009)
#GripTite 293 MSR Cell Line (Invitrogen #R795-07)
#Complete growth media: DMEM (Invitrogen #11965), 10% HIFBS, 0.1mM MEM NEAA, 5mL/L pen-strep (of 5000U/mL stock), 600ug/mL Geneticin
#Assay media: DMEM without phenol red (Invitrogen #21063), 5% charcoal-stripped HIFBS (Hyclone #SH30071.03), 0.1mM MEM NEAA
In-house detection reagent: 300mM Tris-HCl, 300mM Trizma base, 300mM MgCl, 1% Triton-X100, 5mM DTT, 500uM Coenzyme A trilithium salt (Sigma #C3019),450uM ATP (Sigma #A7699), 1mM D-luciferin sodium salt monohydrate (Biosynth #L8240)
Control compounds used were two known firefly luciferase inhibitors (3-(5-(3-chlorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid and 5-(2-fluorophenyl)-3-phenyl-1,2,4-oxadiazole), and DMSO.
Transient transfection of FlucUGA construct: Day 1: GripTite cells were plated in T175 flasks at a density of 1.75 x 107 cells/T175 flask in complete growth media without Geneticin and allowed to grow ON in a 37 degrees Celsius incubator. Day 2: Cells in T175 flask were transfected - with 10ug of DNA and 30uL Transfast in 12mL complete growth media without serum and without Geneticin for 4 hours at 37 degrees Celsius. After incubation, cells were trypsinized, spun down, and resuspended to a concentration of 3.3 x 105 cells/mL in assay media. 6uL of cells in assay buffer were dispensed at 2000cells/well (3.3 x 105 cells/mL) into Greiner solid-white TC-treated 1536-well plates using a Multidrop Combi (Thermo Fisher Scientific). Plates were incubated for one hour at 37 degree Celsius, 95% humidity, 5% CO2. These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 12-point titration of each compound to the assay plate, with a final compound concentration ranging from approximately ~38uM to 0.2nM. Assay plates were then incubated for 48 hours. After incubation, plates were washed three times with DPBS (7uL/well). After the third wash, 6uL of DPBS was added to each well. 3uL of 3X luciferase detection reagent was then added to each well using a flying reagent dispenser (FRD). After a 15 minute incubation to allow for cell lysis, luciferase activity was measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-12 seconds.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)