Journal
BIOORGANIC CHEMISTRY
Volume 94, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bioorg.2019.103395
Keywords
Firefly luciferase; Bioluminescence; Assay interference; Enzyme inhibitor; Inhibition mechanism; Enzyme kinetics; Photoaffinity probe; Enzyme mechanism; Duchenne muscular dystrophy
Funding
- Engineering and Physical Sciences Research Council (EPSRC)
- Medical Research Council (MRC) [EP/L016044/1]
- Summit Therapeutics
- Muscular Dystrophy UK [RA4/3013/4]
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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 (K-D of 0.40 +/- 0.15 mu M). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NF kappa BAI4 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.
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