Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

Yukiya Sako,Masafumi Matsuo,Suguru Yoshida,Takamitsu Hosoya,Masayasu Toyomoto,Atsushi Nishida,Kensuke Ninomiya,Naohiro Hashimoto,Yukiko Okuno,Masatoshi Hagiwara,Isao Kii,Yuka Koike,Kenji Ohe

doi:10.1038/srep46126

Abstract

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. In our previous study, we reported TG003, an inhibitor of CDC2-like kinase 1 (CLK1), as a splice-modifying compound for exon-skipping therapy; however, its metabolically unstable feature hinders clinical application. Here, we show an orally available inhibitor of CLK1, named TG693, which promoted the skipping of the endogenous mutated exon 31 in DMD patient-derived cells and increased the production of the functional exon 31-skipped dystrophin protein. Oral administration of TG693 to mice inhibited the phosphorylation of serine/arginine-rich proteins, which are the substrates of CLK1, and modulated pre-mRNA splicing in the skeletal muscle. Thus, TG693 is a splicing modulator for the mutated exon 31 of the dystrophin gene in vivo, possibly possessing therapeutic potential for DMD patients.

Highlights

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease
We identified an orally available selective CDC2-like kinase 1 (CLK1) inhibitor, TG693, which promoted exon skipping and production of a truncated functional dystrophin protein in immortalized cells derived from a patient with DMD (c.4303G >T) to the same extent as TG003
To test the in vivo stability of TG003 and TG693, mice were administered a single subcutaneous injection of either compound and serum concentrations were monitored by liquid chromatography-mass spectrometry (LC/ MS) at serial time points

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. We previously reported on the small molecule TG003 that promotes skipping of dystrophin exon 31 in cells harboring the c.4303G >T mutation[8]. We identified an orally available selective CLK1 inhibitor, TG693, which promoted exon skipping and production of a truncated functional dystrophin protein in immortalized cells derived from a patient with DMD (c.4303G >T) to the same extent as TG003.

Results

Conclusion