In vitro investigation of the toxicological mechanisms of Fingolimod (S)-phosphate in HEPG2 cells

Fingolimod (FTY720) was the first sphingosine-1-phosphate (S1P) receptor modulator approved by the US Food and Drug Administration for the treatment of multiple sclerosis. The active form, FTY720 (S)-P, acts as a potent agonist of the S1P receptor, leading to its downregulation on the cell surface, reduced activity, and termination of sphingosine-dependent intracellular signalling. Elevated hepatic enzyme levels, clinically significant liver injury, and acute liver failure have been observed in patients treated with FTY720 (S)-P, which requires additional monitoring. This is the first study to investigate the mechanisms underlying the hepatotoxicity of FTY720 (S)-P and represents an important contribution to elucidating its toxicity mechanisms in the human hepatocellular carcinoma cell line HepG2. Following a 72-h exposure, standard methods were used to evaluate specific targets, including cytotoxic effect potentials, mitochondrial parameters, and changes of the antioxidant enzyme levels. FTY720 (S)-P exposure resulted in time- and dose-dependent decreases in cell viability, mitochondrial membrane potential, and ATP levels, as well as the induction of oxidative stress. The complex toxic profile observed for FTY720 (S)-P is hypothesized to originate from its interaction with sirtuin proteins, particularly SIRT3 and SIRT5. It was also complemented with molecular docking simulations to assess the compound's targeting potential by analysing its interaction profile and binding pose within the active sites of both proteins. The results supported the proposed hypothesis, demonstrating an optimal fitting profile and favourable interaction behaviour within the binding pockets of the SIRT3 and SIRT5 enzymes.