Evaluation of Readthrough Efficiency of Negamycin Derivatives against Nonsense Mutations in Muscular Dystrophy Genes

Recently, "readthrough compounds" have attracted attention as a promising approach to treat human hereditary diseases caused by nonsense mutations. These compounds enable ribosomes to bypass a premature termination codon (PTC) introduced into mRNA by a nonsense mutation, thereby restoring the expression of full-length functional proteins. We performed a structure-activity relationship study focusing on (+)-negamycin, a known readthrough compound, and identified potent derivatives, TCP-304 and TCP-306, featuring a cyclopropane moiety. In this study, we investigated how the nature of PTCs and their surrounding nucleotide sequences influence the readthrough activity of these negamycin derivatives, using nonsense mutation sequences derived from Duchenne muscular dystrophy and congenital muscular dystrophy genes. In cell-based reporter assay systems, TCP-306 exhibited potent readthrough efficiency against several nonsense mutation sequences containing the TGA-A. Moreover, its sequence preference differed from that of the aminoglycoside G418, a representative readthrough compound that preferentially induces readthrough at TGA-C sequences, suggesting that TCP-306 may serve as an alternative therapeutic option for muscular dystrophies associated with TGA-A nonsense mutations. Overall, this study provides valuable insights for the development of readthrough drugs for hereditary diseases such as muscular dystrophy caused by nonsense mutations.