Mechanism of action studies of the utrophin modulator ezutromid for the treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disease arising from mutations in the dystrophin gene, affecting about 1 in 5000 boys. Whilst there is currently no available cure for DMD, a number of therapeutic strategies are under development. One such therapy aims to promote expression of utrophin, an autosomal paralogue of dystrophin. Utrophin upregulation rescues the dystrophic phenotype in disease model mice. A novel small molecule utrophin modulator, ezutromid (Summit Therapeutics; formerly SMT C1100), which was identified using a phenotypic screen, progressed to Phase II clinical trials in DMD patients. Interim 24-week data demonstrated reduced muscle fibre damage and increased levels of utrophin, providing the first evidence of ezutromid target engagement and proof of mechanism. However, these effects were not seen after the full 48 weeks of the trial. This work aims to define the mechanism of ezutromid in order to help understand the trial results, and to aid development of new generations of utrophin modulators. Target identification through affinity based protein profiling (AfBPP) has been carried out in this work alongside phenotypic profiling studies. AfBPP requires bioactive analogues suitable for affinity purification. Biotinylated and photoaffinity-labelled analogues of ezutromid designed to be positive and negative control probes have been synthesised and bioactivity confirmed. These probes were used in AfBPP experiments with disease model mouse myoblasts followed by LC-MS/MS. The results from this experiment and phenotype profiling converged on the aryl hydrocarbon receptor (AhR) as a target of ezutromid. Target validation studies demonstrated that ezutromid binds AhR potently, behaving as an AhR antagonist. Moreover, reported AhR antagonists also upregulate utrophin, showing that this pathway could be exploited in future DMD therapies.