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Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. Previous studies have shown that the use of small molecule drugs could be an efficient method to increase the efficacy of delivery of oligonucleotides by cell-penetrating peptides either as targeting agents that can be used in formulation with the cell-penetrating peptide and its cargo or as cell signaling modulators that facilitates the cellular uptake of the treatment. This study presents two aims. The first aim is the identification of small molecule drugs that would induce a synergic effect on the transfection of splice correcting oligonucleotides assisted by PepFect14. The second aim is to identify the mechanisms behind the effect of small molecule drugs modulation of cell-penetrating peptide assisted transfection of oligonucleotides. Through an optimized, high-throughput luciferase assay for short oligonucleotide delivery using cell-penetrating peptides, and the simultaneous addition of a small molecule drug library, we show that three small molecule drugs (MPEP, VU0357121 and Ciproxifan) induced an increase in the transfection efficacy of PepFect14 in complex with a short single-stranded oligonucleotide in HeLa pLuc705 cells. These three drugs are described in the literature to be highly specific for their respective target receptors. However, none of those receptors are expressed in our cell line, indicating a yet non-described pathway of action for these small molecules. We show that the indicated small molecules, without interfering with the particles formed by PepFect14 and the oligonucleotide, interfere via still unidentified interactions in cell signaling, leading to an up-regulation of endocytosis and a higher efficacy in the delivery of short splice correcting oligonucleotides in complex with PepFect14., Competing Interests: The authors received funding in part from the European Federation of Pharmaceutical Industries and Associations, EFPIA. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development, or marketed products to declare.

In this study, through the use of protein mimicry, a peptide was developed to activate the dopamine 1 receptor signaling pathway from the inside of the cell and in absence of the natural extracellular ligand. The sequence was initially derived from the intracellular interaction site between the activated receptor and the alpha domain of its associated G-protein and subsequently modified to increase its cell-penetrating properties. The peptide was then synthesized via solid phase peptide synthesis, purified and tested on cell models. This novel lipopeptide proved to be capable of efficiently ubiquitously penetrating the cell without the need for transfection agents or chiral recognition by specific pathways. Furthermore, the peptide induced the cellular response normally achieved through the activation of the receptor in cells that had not been treated with the natural ligand. The peptide could work as a candidate substitute to l-DOPA, leading the way for a peptides-based treatment for Parkinson's disease. [ABSTRACT FROM AUTHOR]