AID	Name	Type	Comment
2309	Probe Summary for Inhibitors and Stabilizers of Firefly Luciferase	summary	summary AID

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 antagonists of miR-21. Compounds that inhibit miR-21 or compounds that stabilize FLuc intracellularly are identified as active compounds in the assay.

NCGC Assay Protocol Summary:

Reagents: 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)
HeLa cells stably expressing firefly luciferase (FLuc) under the control of a CMV promoter (upstream) and a miR-21 binding sequence directly downstream of the gene. These cells endogenously express miR-21.
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.

Assay Summary:
6uL of HeLa miR-21-FLuc cells in assay buffer (DMEM, Invitrogen #21063-029; 10% FBS, HyClone #SH30071.03) 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 allowed to come to RT for one hour prior to delivery of 3uL of 3X luciferase detection reagent. 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.

Compound Ranking:

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.