Dual-action mitochondria-targeted prodrugs that both deplete mitochondrial glutathione and deliver a toxic payload to the matrix

Mitochondrial glutathione (mGSH) protects the organelle and the cell against reactive oxygen species (ROS), electrophilic metabolites and xenobiotics. Many cancers upregulate GSH to confer resistance against cell death by ferroptosis and anticancer drugs, so mGSH depletion is a potential anticancer strategy. We previously developed MitoCDNB, a mitochondria-targeted molecule that selectively depletes mGSH and disrupts mitochondrial thiol redox homeostasis. However, mGSH depletion by MitoCDNB required catalysis by glutathione-S-transferases (GSTs). Here, we develop a dual-action prodrug scaffold to deplete mGSH independently of GSTs and simultaneously release a payload to increase oxidative stress. The scaffold has four components: a triphenylphosphonium (TPP) group for targeting to the mitochondria, a GSH-reactive electrophilic dinitroaryl ring bearing a sulfonamide leaving group for depleting mGSH, an ethylenediamine-derived self-immolative linker and a phenolic payload. The rates of nucleophilic aromatic substitution (S N Ar) of the sulfonamide by GSH and the cyclisation of the released linker-payload intermediate were measured and the kinetics successfully modelled as consecutive reactions. Under physiological levels of GSH (10 mM) and matrix pH (8.0), our best linker releases a 7-hydroxycoumarin reporter with a half-life of 2.5 min at 30 °C. We used the scaffold for cellular and mitochondrial uptake of a compound that depletes mGSH and releases the redox-cycling pro-oxidant, menadiol/menadione, in the mitochondrial matrix. The combination of mGSH depletion with enhanced mitochondrial ROS production showed synergistic cytotoxicity towards cancer cells, paving the way for the development of dual-action mitochondria-targeted prodrugs as potential cancer therapeutics. • Dual-action prodrugs are activated by glutathione to release a phenolic payload. • The prodrugs deplete glutathione in mitochondria via formation of a conjugate. • The prodrugs selectively deliver the payload to the mitochondria in cells. • This system delivers the ROS-generating drug, menadiol, to mitochondria. • Glutathione depletion and ROS generation showed synergistic cytotoxicity.