Triphenylphosphonium Moiety Modulates Proteolytic Stability and Potentiates Neuroprotective Activity of Antioxidant Tetrapeptides in Vitro

Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP) cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with l-arginine (YRFK) and d-arginine (YrFK) were synthesized and coupled with carboxyethyl-TPP (TPP-3) and carboxypentyl-TPP (TPP-6) units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, l-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.

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The tripeptide glutathione (γ-Glu-Cys-Gly, GSH) and histidine-containing dipeptides, namely carnosine (β-Ala-His) and its homologs, are predominant in mammals and the most studied oligopeptides with intrinsic ROS scavenging and antioxidative functions. Arginine-related bicyclic guanidinium oligomer, which is characterized by enhanced intracellular and intramitochondrial accumulation, was previously developed. Recently, a series of D-tetrapeptides containing two tyrosine residues were synthesized, and N-terminally trifluoroacetylated Tyr-Tyr-His-Pro-HN2 and Tyr-Tyr-Pro-His-NH2 sequence motifs were shown to have highest ability to scavenge ROS and inhibit membrane lipid peroxidation, which was comparable to that of Trolox.

Bioinspired artificial tetrapeptides comprising alternating aromatic (tyrosine, phenylalanine) and cationic (arginine, lysine) amino acids, called Szeto–Schiller (SS) peptides, were earlier developed as dermorphin analogs with pronounced scavenging action against different ROS (Schiller et al., 1989Szeto, 2006a). The aromatic-cationic structure of these peptides, i.e., Tyr-DArg-Phe-Lys-NH2 (starting sequence) and DArg-Dmt-Lys-Phe-NH2 (SS-31 sequence), allows them to pass the plasma membrane and concentrate at the inner membrane of mitochondria (Szeto, 2006a). Cytoprotective and antiapoptotic effects of the SS-peptides were shown to provide therapeutic benefit, for instance, upon exposure to cell-damaging agents (Zhao et al., 2005), ischemia-reperfusion injury (Szeto, 2008), hypertensive cardiomyopathy (Dai et al., 2011), neurodegeneration. (Front Pharmacol. 2018; 9: 115.)