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

Abstract

SUMMARY Firefly 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, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report.

The Ecstasy and Agony of Assay Interference Compounds

“…Over 60 FDA-approved and worldwide drugs contain PAINS chemotypes, 25 and about the same number have been shown to aggregate. 26 Although some drugs can contain PAINS and can aggregate at micromolar concentrations, such examples do not imply that any molecule that acts via a PAINS or aggregation mechanism can become a drug. Hence, noting or "flagging" any PAINS-containing hits and performing detailed follow-up experiments are essential to validate that the function of the molecule is as expected prior to discarding it from further consideration.…”

A Screenable <em>In Vivo</em> Assay for Mitochondrial Modulators Using Transgenic Bioluminescent <em>Caenorhabditis elegans</em>

“…3 One of the compounds resulted in a substantial increase in GFP fluorescence consistent with the inhibitor increasing the levels of GFP-tagged firefly luciferase by binding to the luciferase enzyme's active site, and rendering it more stable. 15 In some instances (not in this particular case) this can lead to increased levels of bioluminescence when the inhibitor is displaced by excess substrate. 15 It is, therefore, important not to interpret results erroneously from compounds that interact with the firefly enzyme as decreased or increased ATP levels/mitochondrial function.…”

“…15 In some instances (not in this particular case) this can lead to increased levels of bioluminescence when the inhibitor is displaced by excess substrate. 15 It is, therefore, important not to interpret results erroneously from compounds that interact with the firefly enzyme as decreased or increased ATP levels/mitochondrial function. Changes in luminescence signal quite often correlate with additional measurements of health such as movement, development and growth.…”

“…An increase in GFP fluorescence, not explained by growth or compound autofluorescence, may also point to an inhibitor. A number of luciferase inhibitors 15 are known and have been entered into PubChem. Therefore in the first instance, it is good practice to consult information on luciferase inhibitors held by PubChem; followed by testing of the compound effects in in vitro assays with the purified enzyme 3 and/or confirmation of effects on mitochondrial function by additional means.…”

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

“…This should be tested for during secondary screening to confirm the activity of the primary screening hits arising from the use of reporter assays. 188,189 Finally, it is critical to remember that while the use of immortalized cell lines may be more suitable to highthroughput screening, these are not the cells that are affected in SMA, which are primarily motor neurons and possibly skeletal muscle. Thus, reporters may not be able to fully recapitulate the proper regulation of SMN expression and function either in central motor neuron synapses or at the NMJ.…”