Hiroko Yamashita

Amphipathic short helix-stabilized peptides with cell-membrane penetrating ability.

We synthesized four types of arginine-based amphipathic nonapeptides, including two homochiral peptides, R-(L-Arg-L-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-1; R=Ac: Ac-1) and R-(D-Arg-D-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: ent-FAM-1; R=Ac: ent-Ac-1); a heterochiral peptide, R-(L-Arg-D-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-2; R=Ac: Ac-2); and a racemic mixture of diastereomeric peptides, R-(rac-Arg-rac-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-3; R=Ac: Ac-3), and then investigated the relationship between their secondary structures and their ability to pass through cell membranes. Peptides 1 and ent-1 formed stable one-handed α-helical structures and were more effective at penetrating HeLa cells than the non-helical peptides 2 and 3.

A Helix-Stabilized Cell-Penetrating Peptide as an Intracellular Delivery Tool

Two types of cationic cyclic α,α‐disubstituted α‐amino acids: Api C2NH2 (which possesses a lysine mimic side chain) and ApiC2Gu (which possesses an arginine mimic side chain), were developed. These amino acids were incorporated into an arginine‐based peptide sequence [(l‐Arg‐l‐Arg‐dAA)3: dAA=Api C2NH2 or ApiC2Gu], and the relationship between the secondary structures of the resulting peptides and their ability to pass through cell membranes was investigated. The peptide containing ApiC2Gu formed a stable α‐helical structure and was more effective at penetrating cells than the nonhelical Arg nonapeptide (R9). Furthermore, the peptide was able to deliver plasmid DNA into various types of cells in a highly efficient manner.

Oligopeptides with Equal Amounts of l- and d-Amino Acids May Prefer a Helix Screw Sense

We investigated the preferred conformations of two nonapeptides, Boc-(L-Leu-D-Leu-Aib)3-OMe (2) and its enantiomer Boc-(D-Leu-L-Leu-Aib)3-OMe (ent-2), four dodecapeptides, Boc-(L-Leu-D-Leu-Aib)4-OMe (3), Boc-(L-Leu-Aib-D-Leu)4-OMe (4), Boc-(Aib-L-Leu-D-Leu)4-OMe (5), and Boc-(L-Leu-Aib-D-Leu-Aib)3-OMe (6), and a decapeptide, Boc-L-Leu-(D-Leu-L-Leu-Aib)3-OMe (7), in solution and in the crystalline state. The nonapeptide 2 formed a right-handed (P) α-helix, and its enantiomer ent-2 formed a left-handed (M) α-helix. The dodecapeptides 3 and 5 were folded into (P) helices, and 4 formed an (M) helical structure. As for 6, roughly equivalent amounts of (P) and (M) helices were observed in solution, and two (M) α-helices were detected in the crystalline state. Furthermore, the decapeptide 7, which possesses four L-Leu residues and three D-Leu residues, was folded into an (M) α-helix.

Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier.

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.

Plasmid DNA delivery using fluorescein-labeled arginine-rich peptides

Arginine (Arg)-rich peptides exhibit an effective cell-penetrating ability and deliver membrane-impermeable compounds into cells. In the present study, three types of Arg-rich peptides, R9 containing nine Arg residues, (RRG)3 containing six Arg and three glycine (Gly) residues, and (RRU)3 containing six Arg and three α-aminoisobutyric acid (Aib) residues, were evaluated for their plasmid DNA (pDNA) delivery and cell-penetrating abilities. The transfection efficiency of R9/pDNA complexes was much higher than those of (RRG)3 and (RRU)3/pDNA complexes, and was derived from the enhanced cellular uptake of R9/pDNA complexes. The replacement of three Arg residues with the neutral amino acid Gly and hydrophobic amino acid Aib drastically changed the cell-penetrating ability and physicochemical properties of peptide/pDNA complexes, resulting in markedly reduced transfection efficiency. A comparison of the R9 peptide administration forms between a peptide alone and peptide/pDNA complex revealed that the uptake of R9 peptides was more efficient for the complex than the peptide alone, but occurred through the same internalization mechanism. The results of the present study will contribute to the design of novel Arg-rich cell-penetrating peptides for pDNA delivery.

Topological Study of the Structures of Heterochiral Peptides Containing Equal Amounts of l-Leu and d-Leu

We designed and synthesized two dodecapeptides, Boc-(l-Leu-l-Leu-Aib-d-Leu-d-Leu-Aib)2-OMe (5) and Boc-l-Leu-l-Leu-Aib-(d-Leu-d-Leu-Aib)2-l-Leu-l-Leu-Aib-OMe (6), that contain equal amounts of l-Leu, d-Leu, and achiral Aib residues. The conformations of peptides 5 and 6 in the crystalline state were studied using X-ray crystallographic analysis. Peptide 5 formed a left-handed (M) α-helical structure, whereas peptide 6 was composed of a combination of fused (M) α-helical and right-handed (P) 310-helical structures. In solution, roughly equivalent amounts of (P) and (M) helices were present in 5, whereas the (M) α-helix was present in 6 as its dominant conformation.

Development of a peptide-based inducer of nuclear receptors degradation.

A peptide-based protein knockdown system for inducing nuclear receptors degradation via the ubiquitin-proteasome system was developed. Specifically, the designed molecules were composed of two biologically active scaffolds: a peptide that binds to the estrogen receptor α (ERα) surface and an MV1 molecule that binds to cellular inhibitors of apoptosis proteins (IAP: cIAP1/cIAP2/XIAP) to induce ubiquitylation of the ERα. The hybrid peptides induced IAP-mediated ubiquitylation followed by proteasomal degradation of the ERα. Those peptides were also applicable for inducing androgen receptor (AR) degradation.

Effects of D-Leu Residues on the Helical Secondary Structures of L-Leu-Based Nonapeptides

The influence of D-Leu residues on the helical structures of L-Leu-based-nonapeptides was investigated. Specifically, the preferred conformations of four diastereomeric nonapeptides, Boc-(L-Leu-L-Leu-Aib)3-OMe (1); Boc-(L-Leu-L-Leu-Aib)2-L-Leu-D-Leu-Aib-OMe (2), which contained one D-Leu residue; Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3), which contained two D-Leu residues; and Boc-(L-Leu-D-Leu-Aib)3-OMe (4), were analyzed in solution and in the crystalline state. Peptide 1 formed a right-handed (P) 310-helix in solution. Peptides 2 and 3 both formed (P) 310-helices in solution and (P) α-helices in the crystalline state. Peptide 4 formed a (P) α-helix both in solution and in the crystalline state.

The side-chain hydroxy groups of a cyclic α,α-disubstituted α-amino acid promote oligopeptide 310 -helix packing in the crystalline state.

A single chiral cyclic α,α‐disubstituted amino acid with side‐chain methoxymethyl (MOM) protecting groups, (3S,4S)−1‐amino‐(3,4‐dimethoxymethoxy)cyclopentanecarboxylic acid [(S, S)‐Ac5cdOMOM], or side‐chain hydroxy groups, (3S,4S)−1‐amino‐(3,4‐dihydroxy)cyclopentanecarboxylic acid [(S, S)‐Ac5cdOH], was attached to the N‐terminal or C‐terminal position of α‐aminoisobutyric acid (Aib) tetrapeptide segments; i.e., we designed and synthesized four pentapeptides, Cbz‐[(S, S)‐Ac5cdOMOM]‐(Aib)4‐OEt (1), Cbz‐[(S, S)‐Ac5cdOH]‐(Aib)4‐OEt (2), Cbz‐(Aib)4‐[(S, S)‐Ac5cdOMOM]‐OMe (3), and Cbz‐(Aib)4‐[(S, S)‐Ac5cdOH]‐OMe (4). We then analyzed the peptides’ structures in the crystalline state. The four peptides all folded into 310‐helical structures; 1 formed a left‐handed (M) 310‐helix, 2 formed a mixture of right‐handed (P) and (M) 310‐helices, 3 formed a mixture of (P) and (M) 310‐helices, and 4 formed a (P) 310‐helix, respectively. In packing mode, the molecules of peptides 1 and 3, which both possessed an Ac5cdOMOM residue, were connected by intermolecular hydrogen bonds along the peptide backbone (NH···O type). On the other hand, the packing of peptides 2 and 4, which both contained an Ac5cdOH residue, was based on intermolecular hydrogen bonds derived from both the peptide backbone and the side‐chain hydroxy groups of the amino acid Ac5cdOH (OH···O type).