Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine
Phosphopeptides are valuable reagent probes for studying protein-protein and protein-ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent labeled phosphopeptides, including F'-GpYLPQTV, F'-NEpYTARQ F'-AEEEIYGEFEAKKKK, F'-PEpYLGLD, F'-pYVNVQN-NH2, and F'-GpYEEI (F' = fluorescein), was evaluated in the presence or absence of a [WR](4), a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR](4) improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F'-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR](4) and was found to be time-dependent. Confocal microscopy of a mixture of F'-PEpYLGLD and [WR](4) in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F'-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR](4). TEM results showed that the mixture of PEpYLGLD and [WR](4) formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR](4) and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR](4) can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides.
Nasrolahi Shirazi, Amir, Rakesh Kumar Tiwari, Donghoon Oh, Antara Banerjee, Arpita Yadav, and Keykavous Parang. "Efficient delivery of cell impermeable phosphopeptides by a cyclic peptide amphiphile containing tryptophan and arginine." Molecular pharmaceutics 10, no. 5 (2013): 2008-2020.
American Chemical Society