Kentaro Takayama

Enhanced intracellular delivery using arginine-rich peptides by the addition of penetration accelerating sequences (Pas)

Cell penetrating peptides (CPPs), including arginine-rich peptides, are attractive tools for the intracellular delivery of various bioactive molecules with a low membrane permeability. We showed that the accelerated intracellular delivery of arginine-rich peptides was achieved by the addition of a short peptide segment (penetration accelerating sequence, Pas) to arginine-rich CPPs. The cytosolic release of the Pas-attached arginine-rich CPPs was observed within 5 min after the treatment of the cells with the peptides even in the presence of serum. Effectiveness of the Pas segment in the intracellular delivery of bioactive peptides using arginine-rich CPPs was exemplified through the enhanced growth inhibition activity of the malignant glioma cells by a retro-inverso peptide derived from the p53 C-terminal 22-amino-acid segment (positions 361-382).

Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA

An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy.

Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD

The capacity of cell penetrating peptides (CPPs) to breach biological membranes offers hope for their utilisation as vectors for the delivery of small molecule drugs and macromolecular therapeutics. Using three different cell systems, including primary human cells, we have studied the uptake, subcellular localisation and effect on cell viability of the well characterised octaarginine and the more recently discovered hydrophobic PFVYLI peptide, either alone, or conjugated to the proapoptotic domain peptide PAD (klaklak)(2). Octaarginine and PFVYLI were efficiently endocytosed into cells at 37 degrees C but an ability to translocate directly across the plasma membrane at higher peptide concentrations or when uptake experiments were performed on ice was confined to the cationic variant. Octaarginine- and PFVYLI-PAD conjugates were cytotoxic, with KG1a leukaemia cells being more sensitive than HeLa cells and octaarginine-PAD being the most potent conjugate in both cell lines. The effects of the CPP-PAD conjugates on cell morphology and permeability was rapid suggesting that cytotoxicity is partially mediated at the plasma membrane rather than exclusively through induction of apoptosis at the mitochondria. Primary human leukaemia cells were more similar to KG1a cells than HeLa cells, suggesting the relative sensitivity of leukaemia cells to these peptides could be exploited in vivo.

Acylation of octaarginine: Implication to the use of intracellular delivery vectors

Cell-penetrating peptides (CPPs) have the ability to efficiently internalize into cells and thus have been used as a vector for the intracellular delivery of various bioactive molecules. The introduction of a hydrophobic core to CPPs may increase their interaction with membranes and facilitate their translocation into cells. While the usefulness of acylated oligoarginine to gene and siRNA delivery has been largely reported, little information is available about their use for the delivery of small molecular-weight compounds, peptides and proteins. In this report, we employed octaarginine (R8) as a typical arginine-rich CPP and evaluated the effect of acylation using butanoic, hexanoic and decanoic acids on its capacity as a delivery vector. Hexanoyl octaarginine (C6R8-Alexa) showed the highest efficiency of cellular uptake of the studied variants, ten times higher than R8-Alexa. C6R8-Alexa also produced a diffuse cytosolic distribution. On the other hand, a less significant effect of C6R8 over R8 was observed for the delivery of proteins, suggesting that the advantage of C6R8 may be obtained during the delivery of relatively small molecular-weight compounds. Although less prominent than at 37°C, a significant cytosolic distribution of C6R8-Alexa was observed at 4°C, and this suggested the potential ability of the C6R8 peptide for direct penetration through plasma membranes.

Allogeneic cardiospheres delivered via percutaneous transendocardial injection increase viable myocardium, decrease scar size, and attenuate cardiac dilatation in porcine ischemic cardiomyopathy

Background Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy. Methods and Results We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled (“dose optimization”) study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled (“pivotal”) study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo. Conclusions Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration.

Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery.

Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained.

Novel System to Achieve One-Pot Modification of Cargo Molecules with Oligoarginine Vectors for Intracellular Delivery

There is a growing number of reports showing the usefulness of cell-penetrating peptides (CPPs) including oligoarginines for intracellular delivery of macromolecules. Although the covalent attachment of the CPP segments to the cargo molecules is usually required to ensure effective delivery, conventional methods of conjugation need several manipulation steps that are often time-consuming and laborious. Here, we report a novel approach to allow easy modification of cargo molecules with oligoarginine CPPs. The key feature is the employment of oligoarginines (R8 and R12) equipped with a sulfosuccinimidylsuberyl moiety (BS(3)-R8 and -R12). One-pot modification is achieved simply by mixing BS(3)-R8 and -R12 with cargos in an aqueous buffer. The usefulness of this approach was exemplified through the conjugate formation with Fab fragments of immunoglobulin G, amino-functionalized poly(ethylene glycol)s (amino-PEGs), and amino quantum dots (amino-QDs), yielding an efficient cellular uptake.

Identification of the Minimum Peptide from Mouse Myostatin Prodomain for Human Myostatin Inhibition

Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice.

Discovery of Selective Hexapeptide Agonists to Human Neuromedin U Receptors Types 1 and 2

Neuromedin U (NMU) are bioactive peptides with a common C-terminal heptapeptide sequence (FLFRPRN-amide, 1a) among mammals, which is responsible for receptor activation, namely NMU receptor types 1 (NMUR1) and 2 (NMUR2). Among the various physiological actions of NMU, the anorexigenic effect has recently attracted attention in drug discovery efforts for treating obesity. Although several structure-activity relationship (SAR) studies have been reported, receptor-selective small peptide agonists have yet to be disclosed. Herein a SAR study of 1a-derived peptide derivatives is described. We initially screened both human NMUR1- and NMUR2-selective peptides in calcium-mobilization assays with cells transiently expressing receptors. Then we performed a precise assay with a stable expression system of receptors and consequently discovered hexapeptides 8d and 6b possessing selective agonist activity toward each respective receptor. Hexapeptide 6b, which selectively activates NMUR2 without significant NMUR1 activation, should aid in the development of anorexigenic drugs as well as advance NMU-related endocrinological research.

Development of an intracellularly acting inhibitory peptide selective for PKN

PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485-499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki=0.7 muM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.