In vivo genotoxicity assessment of selected compounds for the development of HDAC4-targeting PROTACs in spinal muscular atrophy

Progressive motor neuron loss and skeletal muscle atrophy are characteristic features of spinal muscular atrophy (SMA), a severe inherited neuromuscular disease. Despite significant advances in SMA treatment, there is still a need for the development and preclinical characterization of novel therapeutic strategies. Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) represents a promising approach for modulating disease-relevant proteins. In particular, selective degradation of histone deacetylase 4 (HDAC4) has been proposed as a potential strategy for interfering with transcriptional pathways associated with muscle atrophy. Along with their pharmacological activity, the safety profile of newly developed PROTAC molecules represents an essential aspect of their preclinical evaluation. In the present study, the potential genotoxicity of two HDAC4-targeting PROTAC molecules, TG-49 and PJ-653, was investigated in vivo using Wistar rats. DNA damage was evaluated by the alkaline comet assay, which enables sensitive detection of DNA strand breaks and related lesions at the level of individual cells. Animals treated with ethyl methanesulfonate (EMS), a well-established genotoxic agent, were included as a positive control, while an appropriate untreated control group was used for comparison. Neither TG-49 nor PJ-653 induced significant DNA damage in the liver, kidney, or femur samples compared with the control groups, indicating that the investigated compounds did not exhibit detectable genotoxic effects in the evaluated tissues under the applied in vivo conditions. The obtained results contribute to the safety characterization of TG-49 and PJ-653 and provide additional information relevant to their further preclinical development as potential HDAC4-targeting agents for SMA.