qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor Set
FLuc (firefly luciferase) is the prototypical bioluminescent reporter gene from Photinus pyralis. The enzyme catalyzes the oxidation luciferin via luciferin-AMP to produce light. FLuc is used in cell-based reporter gene-based assays for screening small molecule chemical libraries . However, small molecule luciferase inhibitors present in most large chemical libraries are often enriched in the output of high throughput screens using this reporter sometimes complicating interpretation and candidate selection . ..more
BioActive Compounds: 35
Depositor Specified Assays
FLuc (firefly luciferase) is the prototypical bioluminescent reporter gene from Photinus pyralis. The enzyme catalyzes the oxidation luciferin via luciferin-AMP to produce light. FLuc is used in cell-based reporter gene-based assays for screening small molecule chemical libraries . However, small molecule luciferase inhibitors present in most large chemical libraries are often enriched in the output of high throughput screens using this reporter sometimes complicating interpretation and candidate selection .
RLuc is a bioluminescent reporter gene luciferase from Renilla reniformis, also known as the sea pansy. RLuc uses the luciferin substrate coelenterazine. RLuc is often used in combination with the nonhomologous luciferase FLuc (firefly luciferease) as a trasfection or pathway normalization control . Recently, non-homologous reporters have been described for high throughput screening 'coincidence' reporter gene strategies .
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2. Thorne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol 17: 646-657.
3. Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, et al. (2012) Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Biol 19: 1060-1072.
4. Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A (2010) Cell-based assays: fuelling drug discovery. Anal Bioanal Chem 398: 227-238.
5. Cheng KC, Inglese J (2012) A coincidence reporter-gene system for high-throughput screening. Nat Methods 9: 937.
Biochemical firefly luciferase enzyme assay
NCGC Assay Protocol Summary:
Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.
Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 7-point interplate titration of each compound to the assay plates (quantitative HTS), with final compound concentrations ranging from approximately 60uM to 300pM. One microliter of firefly luciferase in 50mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using an Envision plate reader (PerkinElmer), with an average incubation time of 5 min.
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 inhibitors are ranked higher than compounds that showed apparent activation.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)