Molecular Mechanism of Action of Mitochondrial Therapeutic SS-31 (Elamipretide): Membrane Interactions and Effects on Surface Electrostatics

Abstract

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.

Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte

“…It has recently been shown that SS-31 alters surface electrostatic properties of the mitochondrial inner membrane ( Mitchell et al, 2019 ). The consequences of this effect could include alteration of the channel ion gating properties of the ANT, including conformational changes secondary to enhanced supercomplex and ATP synthasome complex stability.…”

“…It is possible that this directly affects stability of the synthasome components. Alternatively, the effect could be due to the change in inner membrane electrostatic properties that has recently been shown to result from incorporation of SS-31 into the inner membrane ( Mitchell et al, 2019 ), as this could indirectly stabilize the ATP synthasome.…”

Elamipretide for Barth syndrome cardiomyopathy: gradual rebuilding of a failed power grid

“…Importantly, the elamipretide effect of improving membrane integrity was even observed when cardiolipin levels in the in vitro system were lowered to levels consistent with those seen in Barth syndrome [58]. Furthermore, the elamipretide-mediated effects on membranes were observed across independent model systems where cardiolipin was oxidized or enriched with monolysocardiolipin, the immature form of cardiolipin found in Barth syndrome [57,59]. These studies suggest that mitochondrial structure and function are, as expected, interdependent and demonstrate that elamipretide targets mitochondrial membranes to stabilize cristae networks and improves bioenergetic function [58].…”

“…1) leads to a restoration of healthy gene expression (within days) and improved cardiac and mitochondrial protein turnover (within weeks) [26,62]. Many mitochondrial functions, including oxidative phosphorylation, are strongly associated with the inner mitochondrial membrane [59]. The abnormal structure and function of the mitochondrial network is reflected by impaired mitochondrial cristae structure and loss of mitochondrial network connectivity.…”

“…Elamipretide has been shown by electron and fluorescence microscopy to restore this architecture in compromised mitochondria, resulting in improved cellular bioenergetics [58]. Studies using Barth syndrome patient-derived cell lines and iPSC-derived, beating cardiomyocytes from pediatric patients with cardiomyopathy showed that elamipretide also restores this architecture in compromised mitochondria, leading to improvements in overall cellular function [59]. Furthermore, in cells engineered using CRISPR to have a TAZ mutation, the decline seen in several different ETC subunits and altered mitochondrial quality control was attenuated after 7 days of elamipretide treatment in vitro [63].…”