A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution.

Methods Development


We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quantitative high-throughput screening (qHTS) was used to determine IC(50)s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC(50) < 10 microM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.


Auld, Douglas S; Zhang, Ya-Qin; Southall, Noel; Rai Bantukallu, Ganesha; Landsman, Marc; MacLure, Jennifer; Langevin, Daniel; Thomas, Craig; Austin, Christopher; Inglese, James;


  • Adenosine Triphosphate/ chemistry
  • Animals
  • Benzothiazoles/ chemistry
  • Binding Sites
  • Enzyme Inhibitors/ chemistry
  • Fireflies/ enzymology
  • Firefly Luciferin/ chemistry
  • High-Throughput Screening Assays
  • Luciferases, Firefly/ antagonists & inhibitors
  • Luciferases, Firefly/ chemistry
  • Luminescent Measurements
  • Models, Molecular
  • Oxadiazoles/ chemistry
  • Quantitative Structure-Activity Relationship

External Links