A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained From Directed Evolution

J Med Chem. 2009 Mar 12;52(5):1450-8. doi: 10.1021/jm8014525.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • 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


  • Benzothiazoles
  • Enzyme Inhibitors
  • Oxadiazoles
  • Firefly Luciferin
  • Adenosine Triphosphate
  • Luciferases, Firefly