The mitochondrion is an important therapeutic target due to its roles in energy metabolism, reactive oxygen species production, and activation of cell death. The connection of these cellular processes with diabetes, obesity, neurodegenerative disorders, and cancer makes this organelle a potential control point for treatment of these diseases. The advancement of mitochondrial medicine will be accelerated by the development of organelle-specific cellular transporters as well as by strategies to enhance the activity of mitochondrially-active drugs. Here, the discovery and development of a new class of mitochondria-specific agents, mitochondria-penetrating peptides (MPPs), is described. These peptides, exemplified by the sequences (FXY)3 and (FXY)4 where FX=cyclohexylalanine and Y=basic arginine or lysine residues, display hydrophobic and cationic residues critical for access to this organelle, and appear to accumulate within cells and mitochondria through membrane potential-dependent mechanisms. Subcellular localization of the peptides was determined by the interplay of hydrophobicity and positive charge, and necessary lipophilicity “thresholds” for access to the mitochondrion. MPPs can be engineered to have minimal mitochondrial disruptive activity and cytotoxicity through minimization of hydrophobicity and length. Furthermore, MPPs appear to accumulate predominantly in the mitochondrial matrix, a feature which places them within an exclusive class of mitochondria-specific transporters and may enable delivery applications for a number of therapeutically-relevant cargoes. Information gained from MPP studies on the physiochemical parameters that drive mitochondrial localization were applied to improve the activity of the anticancer peptide d-(KLAKLAK)2, an agent that activates apoptosis through mitochondrial disruption. Residue substitutions that increased peptide hydrophobicity, regardless of changes to secondary structure, enhanced mitochondrial localization, activity, and cytotoxicity induced by the peptide. In conclusion, these studies provide important guidelines for how to drive the subcellular localization and activity of peptides, and expand the possibilities for mitochondrially-targeted therapeutics.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

The mitochondrion is an important therapeutic target due to its roles in energy metabolism, reactive oxygen species production, and activation of cell death. The connection of these cellular processes with diabetes, obesity, neurodegenerative disorders, and cancer makes this organelle a potential control point for treatment of these diseases. The advancement of mitochondrial medicine will be accelerated by the development of organelle-specific cellular transporters as well as by strategies to enhance the activity of mitochondrially-active drugs. Here, the discovery and development of a new class of mitochondria-specific agents, mitochondria-penetrating peptides (MPPs), is described. These peptides, exemplified by the sequences (FXY)3 and (FXY)4 where FX=cyclohexylalanine and Y=basic arginine or lysine residues, display hydrophobic and cationic residues critical for access to this organelle, and appear to accumulate within cells and mitochondria through membrane potential-dependent mechanisms. Subcellular localization of the peptides was determined by the interplay of hydrophobicity and positive charge, and necessary lipophilicity “thresholds” for access to the mitochondrion. MPPs can be engineered to have minimal mitochondrial disruptive activity and cytotoxicity through minimization of hydrophobicity and length. Furthermore, MPPs appear to accumulate predominantly in the mitochondrial matrix, a feature which places them within an exclusive class of mitochondria-specific transporters and may enable delivery applications for a number of therapeutically-relevant cargoes. Information gained from MPP studies on the physiochemical parameters that drive mitochondrial localization were applied to improve the activity of the anticancer peptide d-(KLAKLAK)2, an agent that activates apoptosis through mitochondrial disruption. Residue substitutions that increased peptide hydrophobicity, regardless of changes to secondary structure, enhanced mitochondrial localization, activity, and cytotoxicity induced by the peptide. In conclusion, these studies provide important guidelines for how to drive the subcellular localization and activity of peptides, and expand the possibilities for mitochondrially-targeted therapeutics.