Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase

Bioorg Chem. 2020 Jan:94:103395. doi: 10.1016/j.bioorg.2019.103395. Epub 2019 Oct 28.

Abstract

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.

Keywords: Assay interference; Bioluminescence; Duchenne muscular dystrophy; Enzyme inhibitor; Enzyme kinetics; Enzyme mechanism; Firefly luciferase; Inhibition mechanism; Photoaffinity probe.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoxazoles / chemical synthesis
  • Benzoxazoles / chemistry
  • Benzoxazoles / pharmacology*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Fireflies / enzymology*
  • Kinetics
  • Luciferases, Firefly / antagonists & inhibitors*
  • Luciferases, Firefly / metabolism
  • Molecular Structure
  • Structure-Activity Relationship

Substances

  • Benzoxazoles
  • Enzyme Inhibitors
  • SMT C1100
  • Luciferases, Firefly