A new triphenylphosphonium-conjugated amphipathic cationic peptide with improved cell-penetrating and ROS-targeting properties

Rezeda A Ishkaeva; Diana V Salakhieva; Ruslan Garifullin; Raghad Alshadidi; Alexander V Laikov; Abdulla A Yergeshov; Marat I Kamalov; Timur I Abdullin

doi:10.1016/j.crphar.2022.100148

A new triphenylphosphonium-conjugated amphipathic cationic peptide with improved cell-penetrating and ROS-targeting properties

Curr Res Pharmacol Drug Discov. 2022 Dec 17:4:100148. doi: 10.1016/j.crphar.2022.100148. eCollection 2023.

Authors

Rezeda A Ishkaeva¹, Diana V Salakhieva¹, Ruslan Garifullin^{1

2}, Raghad Alshadidi¹, Alexander V Laikov¹, Abdulla A Yergeshov¹, Marat I Kamalov¹, Timur I Abdullin¹

Affiliations

¹ Department of Biochemistry, Biotechnology, Pharmacology, Institute of Fundamental Medicine and Biology, Kazan Volga Region Federal University, 18 Kremlyovskaya St., 420008, Kazan, Russia.
² Department of Aeronautical Engineering, University of Turkish Aeronautical Association, Türkkuşu Kampüsü, 06790, Ankara, Turkey.

Abstract

We study for the first time whether triphenylphosphonium (TPP) moiety can improve cellular delivery and redox properties of amphipathic cationic peptides based on YRFK/YrFK cell-penetrating and cytoprotective motif. TPP moiety was found to increase reducing activity of both stereoisomeric peptides in solution and on electrode surface in association with TPP-mediated intramolecular interactions. Among TPP-conjugated peptides, newly synthesized TPP3-YrFK featured both increased antioxidant efficacy and proteolytic resistance. TPP-conjugated peptides preferably mitigated endogenic ROS in mitochondria and cytoplasm of model glioblastoma cells with increased oxidative status. This anti-ROS effect was accompanied by mild reversible decrease of reduced glutathione level in the cells with relatively weak change in glutathione redox forms ratio. Such low interference with cell redox status is in accordance with non-cytotoxic nature of the compounds. Intracellular concentrations of label-free peptides were analyzed by LC-MS/MS, which showed substantial TPP-promoted penetration of YrFK motif across cell plasma membrane. However, according to ΔΨ_m analysis, TPP moiety did not profoundly enhance peptide interaction with mitochondrial inner membrane. Our study clarifies the role of TPP moiety in cellular delivery of amphipathic cationic oligopeptides. The results suggest TPP moiety as a multi-functional modifier for the oligopeptides which is capable of improving cellular pharmacokinetics and antioxidant activity as well as targeting increased ROS levels. The results encourage further investigation of TPP3-YrFK as a peptide antioxidant with multiple benefits.

Keywords: ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); Amphipathic cationic peptides; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CD, circular dichroism; Cellular pharmacokinetics; DCFDA, 2′,7′-dichlorofluorescin diacetate; GSH, reduced glutathione; HBSS, Hank's balanced salt solution; Intramolecular interaction; LC–MS/MS, liquid chromatography tandem mass-spectrometry; MCB, monochlorobimane; MRM, multiple reaction monitoring; ROS targeting; ROS, reactive oxygen species; Redox activity; SPPS, solid-phase peptide synthesis; TPP, triphenylphosphonium; Triphenylphosphonium cation; aa, amino acid.