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.

Highlights

  • Luciferase (FLuc) is widely used in academia and industry due to its excellent sensitivity and dynamic range, and its ease of use [1]

  • SMT C1100 was originally discovered using a Firefly luciferase (FLuc) reporter gene assay for utrophin upregulation

  • It was later shown to upregulate utrophin at both the mRNA and protein level in Duchenne muscular dystrophy (DMD) mouse models and in human DMD patients’ cells [42,43,44], in dose-response analyses in the original reporter gene assay it displayed a bell-shaped curve, distinctive of FLuc inhibitors which stabilise FLuc and inhibit the bioluminescence reaction at high concentrations (Fig. 2a). This led us to suspect that an interaction between ezutromid and FLuc may be at least contributing to the output

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Summary

Introduction

Luciferase (FLuc) is widely used in academia and industry due to its excellent sensitivity and dynamic range, and its ease of use [1]. Compound binding has been found to impart a thermal stabilisation on the FLuc enzyme, which increases its half-life independently of transcription or translation [3,12]. This stabilisation effect is amplified by the short half-life of FLuc coupled with long compound incubation times (12–48 h) typically seen with reporter gene assays [13]. Increases in luciferase abundance by compound binding can lead to false positives, while false negatives arise from inhibition of the bioluminescence reaction. FLuc can perform ‘dark’ reactions such as oxidation of the intermediate LH2AMP to form the potent tight-binding inhibitor L-AMP 5 which is responsible for the flashing of firefly lanterns [11,14,17]

Mechanisms of FLuc inhibition
SMT C1100 is an FLuc inhibitor
SMT C1100 does not compete with luciferin or ATP
Design and synthesis of a photoaffinity probe
Oxidation of probe reveals an alternative mechanism
Conclusions
Materials
Cell culture
FLuc fluorescence quenching assay
Biochemical FLuc inhibition assay
Docking of FLuc luciferin pocket
FLuc diazirine irradiation study
Full Text
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