Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto–Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.

SignificanceSzeto-Schiller (SS) peptides are among the most promising therapeutic compounds for mitochondrial dysfunction. However, the molecular target(s) and the mechanism of action of SS peptides are poorly understood. In this study, we evaluate the interaction of the lead compound SS-31 (Elamipretide) with mitochondrial and synthetic model membranes using a host of biophysical techniques. Our results show that SS-31 membrane interaction is driven largely by the negative surface charge of mitochondrial membranes and that SS-31 alters lipid bilayer properties, most notably electrostatics at the membrane interface. This work supports a mechanism in which SS peptides act on a key physical property of mitochondrial membranes rather than with a specific protein complex, consistent with the exceptionally broad therapeutic efficacy of these compounds. AbstractMitochondrial dysfunction includes heritable diseases, acquired pathologies, and age-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that have demonstrated efficacy in treating a wide array of mitochondrial disorders, and are believed to target mitochondrial membranes due to their enrichment in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, we have analyzed the interactions of the lead compound SS-31 (Elamipretide) with model and mitochondrial membranes using biophysical and computational approaches. Our results show that this polybasic peptide partitions into the membrane interfacial region with affinity and binding density that are directly related to surface charge. SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it does cause saturable alterations in lipid packing. Most notably, SS-31 modulates the surface electrostatic properties of model and mitochondrial membranes, which could play a significant role in the mitoprotective properties of this compound. As a proof of concept, we show that SS-31 alters ion distribution at the membrane interface with implications for maintaining mitochondrial membranes subject to divalent cation (calcium) stress. Taken together, these results support a mechanism of action in which SS peptides interact with lipid bilayers and alter the biophysical (primarily electrostatic) properties of mitochondrial membranes as their primary mechanism of action. Understanding this molecular mechanism is key to the development of future compound variants with enhanced efficacy.

2,4 dinitrophenol (DNP) is an artificial uncoupler of oxidative phosphorylation in mitochondria and was used in therapy against obesity in the mid-1930s. Due to severe side effects and even death cases DNP was prohibited for the therapeutic applications. A renewed interest for DNP originates from the intent to re-use it in small doses for the treatment of obesity, diabetes, hepatic steatosis and neuronal dysfunction. However, several aspects of its action mechanism in mitochondria are not understood. Considering an important role of membrane lipid composition for the mitochondrial uncoupling 1 , we compared the uncoupling effect of DNP in bilayer lipid membranes composed of (i) 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cardiolipin (CL), which mimic inner mitochondrial membrane and (ii) DOPE-free membranes (DOPCþCL). Measurements of total membrane conductance, G, and membrane order parameter, S, revealed that DNP decreases G in concentration-dependent manner in DOPE-containing membranes. In contrast, S was more affected by DNP in DOPC-membranes. MD simulations revealed that (i) DNP-anions are localized in lipid headgroup region whereas protonated DNP were found to be shifted to the membrane centre as shown previously for fatty acids 2 , (ii) maxima of number density profiles for DNP-anion and DOPE overlap and (iii) the average distance between DNP-anion and DOPE headgroup corresponds to those of hydrogen bond. The molecular mechanism is discussed.