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Many reports have shown that stabilization of secondary structure by stapling functional peptides enhances the intracellular bioactivity. However, no report has discussed the correlation between stabilization and biological activity based on the configuration of amino acid residues used as anchors for stapling. To clarify this, we investigated the helix content and apoptotic efficiency of an apoptosis-inducing peptide, Bim, and four stapled Bim peptides containing stapling-related Cys residues introduced with different configurations within the sequence. The results demonstrated that the configuration of Cys residues in stapled Bim peptides affected the secondary structure and intracellular activity of the peptides, and furthermore, there was a correlation between these latter two variables., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Immune checkpoint inhibitors (ICIs) are effective against many advanced malignancies. However, many patients are nonresponders to immunotherapy, and overcoming this resistance to treatment is important. Boron neutron capture therapy (BNCT) is a local chemoradiation therapy with the combination of boron drugs that accumulate selectively in cancer and the neutron irradiation of the cancer site. Here, we report the first boron neutron immunotherapy (B-NIT), combining BNCT and ICI immunotherapy, which was performed on a radioresistant and immunotherapy-resistant advanced-stage B16F10 melanoma mouse model. The BNCT group showed localized tumor suppression, but the anti-PD-1 antibody immunotherapy group did not show tumor suppression. Only the B-NIT group showed strong tumor growth inhibition at both BNCT-treated and shielded distant sites. Intratumoral CD8+ T-cell infiltration and serum high mobility group box 1 (HMGB1) levels were higher in the B-NIT group. Analysis of CD8 + T cells in tumor-infiltrating lymphocytes (TILs) showed that CD62L- CD44 + effector memory T cells and CD69 + early-activated T cells were predominantly increased in the B-NIT group. Administration of CD8-depleting mAb to the B-NIT group completely suppressed the augmented therapeutic effects. This indicated that B-NIT has a potent immune-induced abscopal effect, directly destroying tumors with BNCT, inducing antigen-spreading effects, and protecting normal tissue. B-NIT, immunotherapy combined with BNCT, is the first treatment to overcome immunotherapy resistance in malignant melanoma. In the future, as its therapeutic efficacy is demonstrated not only in melanoma but also in other immunotherapy-resistant malignancies, B-NIT can become a new treatment candidate for advanced-stage cancers., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Multidisciplinary therapy centered on radical surgery for resectable pancreatic cancer is expected to prolong prognosis, but relies on CA19-9 biomarker levels to determine treatment strategy. Boron neutron capture therapy (BNCT) is a chemoradiotherapy using tumor hyperaccumulator boron drugs and neutron irradiation. The purpose of this study is to investigate novel boron drug agents for BNCT for pancreatic cancer. Bioinformatics was used to evaluate the uptake of current boron amino acid (BPA) drugs for BNCT into pancreatic cancer. The expression of the amino acid transporter LAT1, a BPA uptake transporter, was low in pancreatic cancer and even lower in high CA19-9 pancreatic cancer. In contrast, the glucose transporter was high in high CA19-9 pancreatic cancers and inversely correlated with LAT1 expression. Considering the low EPR effect in pancreatic cancer, we synthesized a small molecule Glucose-BSH, which is boron BSH bound to glucose, and confirmed its specific uptake in pancreatic cancer. uptake of Glucose-BSH was confirmed in an environment compatible with the tumor microenvironment. The therapeutic efficacy and safety of Glucose-BSH by therapeutic neutron irradiation were confirmed with BNCT. We report Glucose-BSH boron drug discovery study of a Precision Medicine BNCT with application to high CA19-9 pancreatic cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All of author have no C.O.I., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system.

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.

Although the Omicron variant of the SARS-CoV-2 virus shows resistance to neutralizing antibody, it retains susceptibility to the cellular immune response. Here we characterize vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8 + T cells that strongly suppress Omicron BA.1 replication in vitro. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication is observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation is lost when target cells are treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These ex vivo analysis and in vitro results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell recognition towards emerging variants., (© 2022. The Author(s).)

We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A heterodimeric leucine zipper composed of a pair of leucine zipper peptides containing acidic or basic amino acid residues at appropriate positions in each peptide was used as a molecular glue to connect protein cargos to a cell-penetrating peptide (CPP) carrier. To investigate the hybridization properties by fluorescence experiments, we prepared an enhanced green fluorescent protein (EGFP) fused with an acidic leucine zipper (LzK), EGFP-LzK, and a basic leucine zipper (LzE) modified with a CPP, LzE-CPP. The LzK and LzE formed a 1:1 hybrid when EGFP-LzK and LzE-CPP were mixed in phosphate buffer saline, thereby conjugating the EGFP with the CPP. The formation of the 1:1 hybrid was confirmed by fluorescence spectra and fluorescence titration curves. Results from fluorescence microscopy experiments showed that EGFP was successfully delivered into cells by conjugating with the CPP via formation of the LzK/LzE hybrid. We also fused the apoptotic protein p53 with LzK (p53-LzK) and investigated the inhibition of cell proliferation of various cell lines by incubation with the p53-LzK/LzE-CPP hybrid. This hybrid was found to localize in nuclei and successfully inhibited cell-specific proliferation. The LzE/LzK zipper system inhibited cell proliferation more efficiently than the directly fused conjugate, p53-CPP. Our method will be a useful drug delivery system for delivering bioactive proteins to treat various diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10 B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies-determined by electron microscopy-and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G 1 , S, and G 2 /M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G 1 /S transition than in the G 1 and S/G 2 /M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0-20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen ( 1 O 2 ) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.

Chiral oligopeptide-naphthalene/Eu(iii) hybridized luminophores emit strong circularly polarised solution-state luminescence (CPL) from Eu(iii) at 592 and 614 nm (| g CPL | ≤ 2.1 × 10 -2 ). Although the peptide ligands have matching absolute configurations, the CPL sign is controllable by varying the number of naphthalene units and peptide/Eu(iii) coordination ratio., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The present study investigated combined biological effects of peptide and miRNA in a peptide/miRNA nanocomplex. We utilized TatBim peptide as a cell-penetrating peptide-based RNA carrier with apoptotic activity. miRNA with apoptotic activity (miR-34a) was used for complex formation to investigate the additional effects of the combination with TatBim peptide. TatBim peptide and the miRNA formed nanocomplexes (approximately 250 nm in diameter), and these complexes were efficiently internalized by cells. Despite its efficient cell internalization, apoptotic activity of the nanocomplex decreased with increasing RNA content. However, photosensitizer-attachment to TatBim and photoirradiation significantly improved the apoptotic activity of the nanocomplex by facilitating dispersion of the peptide and RNA in the cytoplasm. Combined apoptotic activity of both TatBim peptide and miR-34a in the nanocomplex was demonstrated by substituting TatBim with Lipofectamine and by substituting miR-34a with scrambled siRNA., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peptide nucleic acids (PNA) are DNA/RNA analogs in which the sugar-phosphate backbone is replaced by N-2-aminoethylglycine. PNA are widely used for experimental antisense therapy due to their strong affinity to mRNA. By targeting specific genes, protein synthesis and the growth of bacteria or cancer cells can be inhibited by PNA. Here, we report the design and evaluation of antisense PNA for selective growth inhibition of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, potent pathogens associated with periodontitis. Antisense PNA against groEL and acpP were prepared with carrier peptide (KFFKFFKFFK). Anti-groEL PNA for P. gingivalis specifically inhibited growth in a dose-dependent manner, and growth was inhibited for 5 h at a concentration of 3 μM. Anti-groEL PNA for A. actinomycetemcomitans inhibited growth for 2 h at a concentration of 3 μM with reduced GroEL protein expression. Anti-acpP PNA did not show a marked growth inhibitory effect on either species. Although further studies are needed to develop more effective antisense PNA for both species, anti-groEL PNA may be potentially useful species-specific antibacterial tools against oral pathogens., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Upon combining chiral peptides (the most basic chiral source) with pyrene moieties, we found that chiral oligopeptides bearing two-pendant pyrenyl units exhibited circularly polarised luminescence (CPL) originating from intramolecular excimers at 450-490 nm in various solvents, and the sign of their CPL signals depended on the type of solvent employed. The CPL and circular dichroism signs and intensities could be tuned by the introduction of a piperidine unit into the chiral peptide chain; thus, the obtained structure could be considered a practical Lock ON-OFF system for oligopeptide luminophores.

Atopic dermatitis is a chronic inflammatory skin disease involving T-helper (Th) 2 cells, eosinophils, and mast cells. Although CCR4 is a major chemokine receptor expressed on Th2 cells and regarded as a potential therapeutic target for allergic diseases, its role in atopic dermatitis remains unclear. Here, by using a hydrogel patch as a transcutaneous delivery device for ovalbumin (an antigen) and Staphylococcus aureus δ-toxin (a mast cell activator), we efficiently induced acute atopic dermatitis-like skin lesions in BALB/c mice, a strain prone to Th2 responses, which were characterized by increased numbers of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions; elevated levels of total and ovalbumin-specific IgE in the sera; and increased expression of IL-4, IL-17A, IL-22, CCL17, CCL22, and CCR4 in the skin lesions. Of note, the same model was less efficient in C57BL/6 mice, a strain prone to Th1 responses. Using this atopic dermatitis model in BALB/c mice, we demonstrated that CCR4-deficiency or a CCR4 antagonist ameliorated the allergic responses. Collectively, these results demonstrate that CCR4 plays a pivotal role in skin allergic inflammation of BALB/c mice by recruiting CCR4-expressing Th2 cells and Th17 cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Multivalent cell-penetrating peptides (CPPs) have been reported to show enhancement in cellular uptake and endosomolytic activity. However, its application was limited to trans-delivery of cargo which is lower in cellular uptake efficiency of cargo than cis-delivery. Here, we tried the cis-delivery of cargo with multivalent CPP by preparing bioreducible dimeric CPP-cargo with apoptotic activity using TatBim peptide, a fusion of Tat CPP and Bim peptide derived from Bim apoptosis-inducing protein. Dimeric TatBim was almost twice as highly internalized by cells and significantly induced apoptosis compared to monomeric TatBim. Contribution of bioreducible linkage of dimeric TatBim towards apoptotic activity was also confirmed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptide-sonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Multiple pyrenes as pendants of enantioimpure di-/tripeptides (abbreviated as N-LD-C, N-DL-C, N-LLD-C and N-DDL-C) showed pyrene-origin CPL and CD signals, which were associated with conflicting CPL-/CD-signs, compared to the corresponding enantiopure di-/tri-peptides.

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Among l- and d-oligopeptides with multiple pyrenes as pendants, the dipeptides with two and three pyrenes showed blue-coloured circularly polarised luminescence as high as |gem|≈ (0.86-1.1) × 10(-2) at around 450 nm, reflecting from exciton couplets of twisted pyrenes.

Glioblastoma, a malignant brain tumor with poor disease outcomes, is managed in modern medicine by multimodality therapy. Boron neutron capture therapy (BNCT) is an encouraging treatment under clinical investigation. In malignant cells, BNCT consists of two major factors: neutron radiation and boron uptake. To increase boron uptake in cells, we created a mercapto-closo-undecahydrododecaborate ([B12HnSH](2-)2Na(+), BSH) fused with a short arginine peptide (1R, 2R, 3R) and checked cellular uptake in vitro and in vivo. In a mouse brain tumor model, only BSH with at least three arginine domains could penetrate cell membranes of glioma cells in vitro and in vivo. Furthermore, to monitor the pharmacokinetic properties of these agents in vivo, we fused BSH and BSH-3R with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA is a metal chelating agent for labeling positron emission tomography (PET) probe with (64)Cu. We administered BSH-DOTA-(64)Cu and BSH-3R-DOTA-(64)Cu to the tumor model through a mouse tail vein and determined the drugs' pharmacokinetics by PET imaging. BSH-3R showed a high uptake in the tumor area on PET imaging. We concluded that BSH-3R is the ideal boron compound for clinical use during BNCT and that in developing this compound for clinical use, the BSH-3R PET probe is essential for pharmacokinetic imaging., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Topical therapy is the most favored form of treatment for whitening against hyper-pigmentation and sunburn because it lends itself to self-administration, patient compliance and an absence of systemic adverse effects. However, high-molecular-weight, hydrophilic chemicals are difficult to use as transdermal delivery drugs and the use of topical drugs has been highly limited. There are now many potent tyrosinase inhibitors, for example, sulfite or kojic acid, but the efficacy of their skin transduction remains a big problem. Furthermore, melanogenesis inhibitors from natural sources have great potential, as they are considered to be safe and largely free from adverse side effects. We applied 11-arginine (11R), a cell-membrane-permeable peptide, as a transdermal delivery system with a skin delivery enhancer, pyrenbutyrate. We performed intracellular screening for melanogenesis inhibitors with 11R fused with several kinds of tyrosinase inhibitory peptides from natural sources. Of 28 tyrosinase peptides, 13 melanin synthesis inhibitory peptides were selected. Peptide No. 10 found in gliadin protein, a wheat component, most strongly inhibited melanin production. This No. 10 peptide, of only 8 amino acids, fused to 11R showed no cytotoxicity and inhibited melanin synthesis as determined through melanin content measured using an absorption spectrometer and observation with a transmission electron microscope. Next, we transduced this 11R-No. 10 into skin with an 11R transdermal delivery system after previous treatment with pyrenbutyrate and performed daily repetitive topical application for two weeks against a UV-induced sun-tanning guinea pig model. We observed a whitening effect in a model skin sample by Masson-Fontana staining and the 11R-No. 10 peptide-applied area showed significant melanogenesis inhibition. These results show that 11R using a transdermal drug delivery system with melanogenesis inhibitory peptide is a very safe and promising method for applications from cosmetics to the pharmaceutical industry., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An indole compound with a strong purple-red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, (13)C NMR, (1)H NMR, UV-visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel(9) 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this report, we describe dendritic peptides possessing central fluorescent amino acids with adjacent branched amino acids. These fluorescent-peptide dendrimers were prepared using (9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis and Fmoc-derivative fluorescent and branched amino acids. The branched amino acids featured multiple carboxylic acids in their side chains, making the fluorescent-peptide dendrimers highly water-soluble compared with the analogous peptides containing the fluorescent amino acids only. The branched amino acid units also improved the fluorescence intensity of the dendrimers. Based on high-pressure liquid chromatography and fluorescence spectroscopy results, we determined that the fluorescent groups were located in the core and that the carboxylic acids were located on the surface of the dendrimers. Fluorescence resonance energy transfer was achieved among the three proximal fluorescent groups in one of the fabricated fluorescent-peptide dendrimers., (Copyright © 2012 Wiley Periodicals, Inc.)

Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor-acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or microscopic polyangiitis.

Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-l-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

EGF receptor-binding peptides could be found by a peptide screening method using fifteen fluorescent amino acids as fluorescent tags. Of 225 peptides, we found an 8-mer peptide containing a dipeptide unit, Y-F, which was the strongest binding peptide to the EGF receptor., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We describe a new screening method for simultaneously detecting peptides that bind to a target protein by fluorescence obtained from fluorescent amino acid-modified peptides.

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridge-builder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.

In this study, an RNA isolation method was developed using a biotinylated peptide nucleic acid (PNA) that is complementary to the target RNA. Using the biotinylated PNA method, we successfully isolated several RNAs from Escherichia coli and from human total RNA in pure form. Damage to the RNA appears to be negligible by this method because the method is rapid and does not require a high temperature treatment to facilitate RNA-PNA binding.

A new series of oxy-peptide nucleic acids (pyrrolidine-based oxy-peptide nucleic acids = POPNAs) of four different stereoisomeric forms (cis-L, cis-D, trans-L, trans-D) have been synthesized. To find a favorable stereoisomer of POPNA for hybridization with DNA or RNA, thermodynamic parameters and conformations of the hybrids between the four stereoisomers with 9 adenine bases [po(A(9))s] and dT(9) or rU(9) were investigated from ultraviolet (UV) melting curves and circular dichroism (CD) spectra. The cis-L-po(A(9)) formed the most stable hybrid with dT(9), because of the smallest entropy loss, despite the smallest enthalpy gain. In contrast, trans-L-po(A(9)) formed the most stable hybrid with rU(9), because of the largest enthalpy gain, despite the largest entropy loss. The hybrid stability of trans-L-po(A(9)) with rU(9) was significantly improved as compared with a previous version of oxy-peptide nucleic acid (OPNA) that lacks the pyrrolidine ring., (Copyright 2006 Wiley Periodicals, Inc.)

A novel method to isolate a single kind of RNA from RNA mixture was developed by using a biotinylated-PNA (peptide nucleic acid) that is complementary to the RNA. Efficient bindings of Escherichia coli tRNA(Leu)CUC were observed to the 9-mer and 12-mer PNAs that are complementary to the 3'-end region of the tRNA. Purified E. coli tRNA(Leu)CUC was obtained from E. coli tRNA mixture.

A thymine monomer of pyrrolidine-based peptide nucleic acid that contains a tertiary amino group in the backbone has been synthesized for use in the synthesis of novel peptide nucleic acid.

Four stereoisomers of oxy-peptide nucleic acids containing ether linkages in the main chain and conformationally-restricted pyrrolidine rings (pyrrolidine-based oxy-PNA = POPNA) were newly synthesized and investigated for binding to DNA. cis-l-POPNA with 9 adenine bases formed the most stable hybrid with dT(9). The POPNA showed high sequence specificity similar to that of the Nielsen-type PNA and sharper melting behavior in hybridization with DNA than the Nielsen-type PNA.

We have developed a new series of oxy-peptide nucleic acids with pyrrolidine rings of four different types of structural isomers (cis L, cis D, trans L, trans D configurations; POPNAs). Melting curves of adenine nanomers of POPNAs in the presence of the complementary DNA showed the highest Tm value for cis L-POPNA, but in the case of RNA, the highest Tm value was observed for trans L-POPNA. Thermodynamic parameters (delta H and delta S) of POPNA/DNA and POPNA/RNA hybridizations were evaluated from concentration dependence of Tm values. The POPNA/DNA and POPNA/RNA hybridizations were governed by entropy and enthalpy terms, respectively.