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Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5–16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.

Metabolism is potently regulated by oxygen as the terminal acceptor of the electron transport chain. Thus, a challenge for capturing the in vivo metabolome of animal tissues is to achieve rapid freezing after dissection-induced loss of perfusion, before the onset of hypoxia-driven metabolomic remodeling. However, the timing of the metabolomic changes elicited by post-dissection freezing delays are not well described. We addressed this problem by carefully and systematically assessing broad, genotype-specific, and 13C isotopologue metabolomic change resulting from post-dissection, ex vivo mouse tissue metabolism. Based on experiments with mouse liver, heart muscle, and skeletal muscle, we show that metabolomic change is rapid and extensive, that both false negative and false positive between genotype differences are induced, and that 13C-isotopologue abundances and enrichment percentages change with post-dissection hypoxia. These findings provide a previously absent, systematic illustration that capturing the in vivo tissue metabolome requires immediate post-dissection freezing.

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