DNA-protective activity of selected compounds in the development of HDAC4-targeting PROTACs for spinal muscular atrophy

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder characterised by insufficient levels of the survival motor neuron (SMN) protein and progressive loss of motor neuron function. The disease is primarily caused by alterations in the SMN1 gene, while the paralogous SMN2 gene provides a limited source of functional SMN protein due to its predominant alternative splicing pattern. Consequently, reduced SMN availability compromises motor neuron maintenance and ultimately leads to progressive muscle weakness and atrophy. Although disease-modifying therapies have substantially improved the clinical management of SMA, the identification and characterization of additional therapeutic approaches remain important research objectives. In that sense, targeted degradation of HDAC4 using PROTAC molecules to switch off the HDAC4-atrogin 1-Murf-1 atrophy-associated transcriptional program could be a rational palliative care approach. However, selective HDAC4 degradation could trigger oxidative DNA damage in skeletal muscle by modulating maladaptive GADD45A/myogenin-associated stress signalling, mitochondrial-redox homeostasis, ROS burden, and DNA-damage response pathways involved in genome maintenance. Therefore, four PROTACs developed as anti-SMA agents, namely TG-49, PJ-594, PJ-653, and PJ-655, confirmed as HDAC4 degraders in Jurkat cells, were evaluated in vitro at concentrations of 25, 50, 100, 200, and 400 μg/mL for their protective potential against oxidative DNA damage. DNA-protective activity was assessed using assays based on hydroxyl- and peroxyl-radical-induced DNA damage. All tested compounds exhibited DNA-protective activity against both hydroxyl- and peroxyl-radical-induced oxidative damage across the entire tested concentration range. These findings demonstrate the DNA-protective potential of the investigated compounds and provide additional in vitro evidence supporting their further biological characterization.