Voltage Sensor Probes (VSPs) as an Efficient Tool to Screen for Inhibitors of Voltage-Gated Sodium Channels

Voltage-gated sodium channels (Nav) represent a therapeutically validated group of targets for the development of antiepileptic drugs, analgesics and antiarrhythmics [1]. However most of the existing drugs acting as Nav blockers suffer from multiple side effects, but the existence of a multigene family of Nav [2] suggests that the identification of new compounds that selectively block Nav isoforms might have better therapeutic efficiency and reduced side effects. Due to their molecular interference with numerous ion channels, alkaloids represent a group of natural products of particular interest. This is the reason why we have evaluated the efficiency of an in-house method to screen a library of isoquinoline alkaloids formerly isolated in our laboratory. Mammalian GH3 cells constitutively expressing Nav where used in conjunction with Voltage Sensor Probes (VSPs), the signals being read on a fluorescence plate reader. Thanks to this technique, we were able to precisely detect Nav channels activators or blockers. Among 62 compounds tested, 5 isoquinolines appeared as potent Nav channels inhibitors. References: 1. Salat, K. et al. (2014) EOID 23:1093-1104 2. Yu, F.H. et al (2003) Genome Biol. 4

Voltage Sensor Probes as an efficient tool to screen for new modulators of voltage-gated sodium channels

Voltage-gated sodium channels (Nav) constitute the molecular targets of clinically used drugs for treatments of various diseases (epilepsies, chronic pain, cardiac arrythmies…) and also of numerous toxins from animals and plants. The development of useful screening assay for the discovery of new ligands in/ from chemicals libraries or animal venoms and either plant extract still represents a challenge of great interest. Here, we used a mammalian GH3 cells that constitutively express at least three different brain Nav channels isoforms (Nav1.1, Nav1.2, Nav1.3 and Nav1.6) in order to identify in a library of in-house natural alkaloids, novel compounds of therapeutical interest. For this screening, we developed a method based on the use of Voltage Sensor Probes (VSPs) that we adapted to detect both activators and blockers of Nav channels. Among 62 compounds tested, 5 isoquinolines appeared as potent Nav channels inhibitors. Other compounds were characterized as specific gating modifier of Nav channels. While most of these alkaloids have been already described in the literature, their abilities to act on Nav channels were unknown. In conclusion, we demonstrated the potency of this novel screening method using VSPs to identify novel ligands of Nav channels of therapeutical interests. References: 1. Salat, K. et al. (2014) EOID 23:1093-1104 2. Yu, F.H. et al (2003) Genome Biol. 4

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

Cell-Penetrating Peptides - Mechanisms of Cellular Uptake and Generation of Delivery Systems

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid deliver

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

Targeted Delivery of a Splice-Switching Oligonucleotide by Cationic Polyplexes of RGD-Oligonucleotide Conjugate

Nanoparticle-based delivery has become an important strategy to advance therapeutic oligonucleotides into clinical reality. Delivery by nanocarriers can enhance access of oligonucleotides to their pharmacological targets within cells; preferably, targeting ligands are incorporated into nanoparticles for targeting oligonucleotides to disease sites, often by conjugation to delivery carriers. In this study, a splice-switching oligonucleotide (SSO) was conjugated to a bivalent RGD peptide, and then, the RGD-SSO conjugate was formulated into polyplexes with a cationic polymer polyethylenimine. The resultant polyplexes of RGD-oligonucleotide conjugate demonstrated dramatic increase in the pharmacological response of splicing correction compared to free RGD-SSO conjugate or the polyplexes of unconjugated SSO, through integrin-mediated endocytosis and rapid endosomal release. This study has shown that coupling a targeting ligand to cargo oligonucleotide can maintain the integrin targeting ability after the peptide-oligonucleotide conjugate is complexed with cationic polymer. Preliminary study also revealed that integrin targeting redirects intracellular trafficking of the polyplexes to caveolar pathway and thereby generates greater effectiveness of the oligonucleotide. This study provides a new platform technology to construct multifunctional delivery systems of therapeutic oligonucleotides