Mithramycin Revisited: Exploring the Mechanism of Toxicity in Failure of a Phase I Clinical Trial

Hepatotoxicity is one of the major hurdles that leads to failure during drug development. In most cases, hepatotoxicity is detected in the late stages of drug development either in animal toxicity studies or clinical trials. However, considerable time, effort and resources are spent by the time a compound that may not be viable as a therapeutic agent has undergone animal studies or clinical trials and then is demonstrated to have hepatotoxic side effects. Moreover, patients may suffer when toxicity is first uncovered in first-in-human trials. Mithramycin A (MTH), which we previously found to be active against Ewing Sarcoma, faced this obstacle and therefore failed phase I clinical trials. We thus sought to characterize the mechanism of hepatotoxicity of MTH so compounds with similar hepatotoxic pathways could be avoided in the future.

Herein, we used HepG2 spheroid cultures to assess the hepatotoxicity of MTH. MTH treated spheroids declined in both size and viability at treatment concentrations 1,000 fold lower than those treated with an FDA approved drug, amiloride, which has low hepatotoxic risk and thus served as a control. We then used chemogenomic screening in a pooled Saccharomyces cerevisiae gene deletion collection to generate a hypothesis on the mechanism of hepatotoxicity of MTH. We found 48 non-essential deletion strains. Next, we tested the hypothesis that MTH exerts its hepatotoxicity by disrupting calcium ion homeostasis of the liver. By using florescent calcium labeling in a 2-D HepG2 culture model, we could detect a noticeable increase in free cytosolic calcium levels just 30 minutes after treatment which continued 24 hours post-treatment. While MTH has previously been reported to bind to DNA as its target mechanism of action, our results suggest that the compound modulates its off-target toxicity via the disruption of calcium homeostasis in the liver.