Evaluation of Phenytoin-derived Compounds for Anti-Arrhythmic Potential

The development of novel phenytoin derivatives offers a promising strategy to address the urgent need for safer and more effective anti-arrhythmic therapies. Cardiac arrhythmias are a major cause of morbidity and mortality, contributing to stroke, sudden cardiac death, and long-term disability, yet current pharmacological options are limited by low efficacy and high toxicity. Phenytoin is a Class IB anti-arrhythmic agent that stabilises the cardiac ryanodine receptor 2 (RyR2) and reduces pathological Ca²⁺ leak from the endoplasmic reticulum, a key cellular mechanism driving arrhythmogenesis. Phenytoin’s hydantoin scaffold is chemically robust and highly amenable to structural modification, providing a strong foundation for the rational design of derivatives with enhanced selectivity and safety. Collaborators at the Monash Institute of Pharmaceutical Sciences developed a library of phenytoin-based compounds, identifying over 100 candidates with potential as safe and effective anti-arrhythmic agents. This project aims to establish the anti-arrhythmic abilities of phenytoin derivatives using the HEK293-RyR2 cell assay for screening by examining the propensity of these cells to undergo Ca²⁺ leak events through the transfected ryanodine receptor, providing a rapid, scalable alternative to conventional human cardiac tissue assays. Six derivatives were screened alongside phenytoin controls, with cells loaded with Fluo-4 AM and perfused under increasing extracellular Ca²⁺ concentrations. The assay, previously validated to detect RyR2-modulating effects, confirmed that phenytoin reduced Ca²⁺ leak. Among the derivatives, four showed minimal effect, while one compound markedly suppressed RyR2-mediated leak, exceeding phenytoin’s effect, and another unexpectedly increased leak, illustrating the channel’s sensitivity to subtle chemical modifications. These results provide early insight into structure–activity relationships and highlight the value of iterative design. Overall, the HEK293-RyR2 platform proved robust, scalable, and suitable for high-throughput screening, supporting iterative collaboration with the Monash team to refine derivatives based on the most promising candidates. This approach lays the foundation for next-generation anti-arrhythmic therapies capable of selectively modulating Ca²⁺ handling and reducing RyR2 leak, addressing a critical unmet clinical need in cardiovascular medicine.