Structure-Activity Relationships in the Design of Mitochondria-Targeted Peptide Therapeutics

Mitochondria play a central role in metabolic homeostasis; hence, dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Mitochondria-targeted tetrapeptides with alternating cationic and aromatic residues, such as SS-31 (Elamipretide), show promise as therapeutic compounds. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs that differed with respect to aromatic side chain composition and sequence register, benchmarked against SS-31. Using NMR and molecular dynamics approaches, we obtained the first structural models for this class of compounds, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, the analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. All peptide analogs preserved survival and energy metabolism more effectively than SS-31 in cell stress models. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register strongly influence the activity of these mitochondria-targeted peptides. Furthermore, this work helps provide a framework for the rational design of next-generation therapeutics with enhanced potency.