Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

SummaryFirefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays, owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes, including FLuc-catalyzed formation of multisubstrate adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays, compounds acting as FLuc inhibitors yield paradoxical luminescence increases, thus data on compounds acquired from FLuc-dependent assays require careful analysis as described here.

Graphical AbstractFigure optionsDownload high-quality image (480 K)Download as PowerPoint slideHighlights
► A representative small molecule library contains 12% firefly luciferase inhibitors
► Competitive, noncompetitive, uncompetitive, and MAI inhibitors were identified
► For FLuc-RGAs 65% of FLuc inhibitors with known MOIs increased the luminescence signal
► Methods to identify FLuc inhibitors that interfere with assay results are described