Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency.

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'-O-methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'-O-MDTM groups should be incorporated. siRNA bearing the 2'-O-MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.

Prodrug-Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment.

Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug-type phosphotriester-modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular-mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding to the fully phosphorothioate-modified AON. In this study, we synthesized several types of PTE ONs containing linear disulfides (linear SS PTE ONs) and evaluated their conversion rates under reducing conditions. From the results, the structural requirements for the conversion of the synthesized linear SS PTE ONs were elucidated. Linear SS PTE ON with promising promoieties showed a nuclease resistance up to 4.8-fold compared to unmodified ON and a cellular uptake by endocytosis without any transfection reagent. In addition, although the knockdown activity of the linear SS PTE gapmer AON is weaker than that of the fully phosphorothioate-modified gapmer AON, the knockdown activity is slightly stronger than that of the cyclic SS PTE gapmer AON. These results suggest that the conversion rates may be related to the expression of the antisense activity.

Rediscovery of 4-Trehalosamine as a Biologically Stable, Mass-Producible, and Chemically Modifiable Trehalose Analog.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed. For further analysis, 4-trehalosamine is selected as it shows high producibility. Compared with trehalose, 4-trehalosamine exhibits better or comparable protective activities and a high buffer capacity around the neutral pH. Another advantage of 4-trehalosamine is its chemical modifiability: simple reactions produce its various derivatives. Labeled probes and detergents are synthesized in one-pot reactions to exemplify the feasibility of their production, and their utility is confirmed for their respective applications. The labeled probes are used for mycobacterial staining. Although the derivative detergents can be effectively used in membrane protein research, long-chain detergents show 1000-3000-fold stronger autophagy-inducing activity in cultured cells than trehalose and are expected to become a drug lead and research reagent. These results indicate that 4-trehalosamine is a useful trehalose substitute for various purposes and a material to produce new useful derivative substances.

Influence of Aib-Containing Amphipathic Helical Chain Length in MAP(Aib)-cRGD as Carrier for siRNA Delivery.

MAP(Aib)-cRGD, which is a conjugate of an α-aminoisobutyric acid (Aib)-containing amphipathic helical peptide [MAP(Aib)] with a αv ß3 integrin binding ligand, cRGD, at the C-terminus of the helical peptide, has been developed for siRNA delivery into cells. In this work, we synthesized three peptides containing 19 (PI), 18 (PII), and 17 (PIII) amino acid residues in the helical peptide, which lack Aib, Leu-Aib, and Lys-Leu-Aib residues present in the C-terminus of the helical peptide of the parent MAP(Aib)-cRGD, respectively. MAP(Aib)-cRGD showed the siRNA delivery into cells and the RNAi effect both in the presence and in the absence of serum in reaction media. In contrast, PI delivered siRNA into cells, and this was followed by the RNAi effect in only serum-free reaction media. On the other hand, siRNA delivery was abolished by the further reduction of the number of residues (PII and PIII) in the C-terminus. Our data indicate that the Aib-containing helical part requires 20 residues in the conjugation of the helical peptide with cRGD for the construction of carrier for siRNA delivery into cells.

Enzymatic formation of consecutive thymine-HgII-thymine base pairs by DNA polymerases.

From the perspective of the preparation of a DNA-based nanowire containing an array of metal ions, DNA-polymerase-catalyzed primer extension reactions were investigated and the formation of up to ten consecutive T-HgII-T base pairs was achieved by the HgII-mediated primer extension reaction in the presence of MnII ions. This enzymatic approach may be one of the promising techniques for preparing a DNA-based metal array.

Effects of metal ions on thermal stabilities of DNA duplexes containing homo- and heterochiral mismatched base pairs: comparison of internal and terminal substitutions.

The effects of metal ions on the stabilities of duplexes containing a D-homochiral and heterochiral mismatched base pairs were studied. In some duplexes containing an internal mismatched base pair, significant stabilization by HgII and AgI ions was observed. While, in duplexes containing a terminal mismatched base pair, only the duplexes containing T-T and LT-T mispairs were significantly stabilized by HgII ions, and the stabilities of the duplexes containing T-T and LT-T mispairs exceeded those of the corresponding homochiral matched duplex. The results suggest that the formation of homo- and heterochiral T-HgII-T base pairs at duplex termini would be useful for the thermal and enzymatic stabilization of DNA-based nanodevice.

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases.

Spectroscopic characterization of AgI -ion-mediated C-AgI -A and C-AgI -T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by AgI ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of AgI ions is in the order C-A≈C-T>C-C, the stabilizing effect of AgI ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to AgI -mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.

α-Aminoisobutyric Acid-Containing Amphipathic Helical Peptide-Cyclic RGD Conjugation as a Potential Drug Delivery System for MicroRNA Replacement Therapy in Vitro.

Replacement therapy with tumor suppressive microRNA (TS-miRNA) might be the next-generation oligonucleotide therapy; however, a novel drug delivery system (DDS) is required. Recently, we developed the cell-penetrating peptide, model amphipathic peptide with α-aminoisobutyric acid (MAP(Aib)), as a carrier for oligonucleotide delivery to cells. In this study, we examined whether a modified MAP(Aib) analogue, MAP(Aib)-cRGD, could be a DDS for TS-miRNA replacement therapy. MIR145-5p, a representative TS-miRNA especially in colorectal cancer, was selected. The MAP(Aib)-cRGD dose was adjusted for MIR145-5p delivery to cells using peripheral blood mononuclear cells and degradation analysis. AlexaFluor488-labeled MIR145-5p incorporation into cells and negative regulation of MIR145-5p-targeting genes demonstrated MAP(Aib)-cRGD's functionality as a miRNA DDS. Treating MIR145-5p with MAP(Aib)-cRGD also revealed various anticancer effects, such as cell viability, invasion inhibition, and apoptosis induction in WiDr cells. Altogether, these findings suggest that MAP(Aib)-cRGD could be a DDS for TS-miRNA replacement therapy, but in vivo investigations are required.

Influence of lysine residue in amphipathic helical peptides on targeted delivery of RNA into cancer cells.

The cRGD-conjugated Aib-containing amphipathic helical peptide, MAP(Aib) derivative (PI), has been reported to be a useful carrier for siRNA delivery into cells. We have conducted a series of structure-activity relationship studies of the influence of the balance between hydrophobicity and basicity on the amphipathicity of PI, and synthesized peptides having a larger number of Lys residues than PI. Increasing the number of basic residues in the amphipathic helix suppressed the ability to deliver siRNA into cells. It was concluded that the balance between hydrophobicity and basicity in the PI helix was important for siRNA delivery into cells. Furthermore, the siRNA delivering ability of PI was specific to cancer cells, such as A549, U-87 MG, and WiDr cells, and was low in normal cells, namely, NIH3T3 cells. Next, we examined the potential of PI as a carrier for the delivery of microRNA-133b (miR-133b), which is known to be an anti-oncomiR. PI enhanced the delivery of miR-133b into WiDr cells, which resulted in the suppression of endogenous protein expression.

Correction: Base recognition by l-nucleotides in heterochiral DNA.

[This corrects the article DOI: 10.1039/C2RA01013E.].

Syntheses of prodrug-type 2'-O-methyldithiomethyl oligonucleotides modified at natural four nucleoside residues and their conversions into natural 2'-hydroxy oligonucleotides under reducing condition.

We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2'-O-methyldithiomethyl (2'-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the preparation of 2'-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing 2'-O-(2,4,6-trimethoxybenzylthiomethyl) (2'-O-TMBTM) adenosine, guanosine, and cytidine were reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2'-O-MDTM oligonucleotides in the same manner as the oligonucleotide bearing 2'-O-TMBTM uridine. Furthermore, the oligonucleotides bearing 2'-O-MDTM adenosine, guanosine, and cytidine were efficiently converted into corresponding natural 2'-hydroxy oligonucleotides under the cytosol-mimetic reducing condition.

Effective gene silencing activity of prodrug-type 2'-O-methyldithiomethyl siRNA compared with non-prodrug-type 2'-O-methyl siRNA.

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2'-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2'-O-modified siRNA activities were often decreased by modification, since the bulky 2'-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2'-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2'-O-MDTM siRNAs modified at the 5'-end side including 5'-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2'-O-methyl (2'-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2'-O-MDTM modifications. Our results indicate that 2'-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.

Synthesis and biological evaluation of histone deacetylase and DNA topoisomerase II-Targeted inhibitors.

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities toward histone deacetylases and Topo II, and their cytotoxicities in cancer cell lines, were evaluated. Among the synthesized hybrid compounds, compound 16b showed the potent HDAC inhibitory activity at a low nanomolar level and exhibited antiproliferative activity toward cancer cell lines including MCF-7 (breast), HCT-116 (colon), and DU-145 (prostate) cancer cells at a low micromolar level. Moreover, compound 16a showed HDAC6-selectivity 20-fold over HDAC1.

Corrigendum: Structures and biological activities of novel 4'-acetylated analogs of chrysomycins A and B.

This corrects the article DOI: 10.1038/ja.2017.99.

Structure-activity relationship study of Aib-containing amphipathic helical peptide-cyclic RGD conjugates as carriers for siRNA delivery.

The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure-activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII, as well as PI, could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells.

Valgamicin C, a novel cyclic depsipeptide containing the unusual amino acid cleonine, and related valgamicins A, T and V produced by Amycolatopsis sp. ML1-hF4.

In the course of optimizing pargamicin A production in Amycolatopsis sp. ML1-hF4, we discovered novel cyclic depsipeptide compounds in the broth and designated them valgamicins A, C, T and V. The structures of these molecules were determined by spectroscopic studies, advanced Marfey's method and X-ray crystal structural analysis. Valgamicin C contains the extremely rare amino acid cleonine. To our knowledge, this is the first report of a cleonine-containing metabolite from a naturally isolated microorganism without any breeding or mutation treatment. None of the valgamicins showed potent antibacterial activity against either Gram-positive or -negative bacteria. Valgamicins A, C and T exhibited moderate cytotoxicity against human tumor cell lines.The Journal of Antibiotics advance online publication, 15 November 2017; doi:10.1038/ja.2017.135.

Structures and biological activities of novel 4'-acetylated analogs of chrysomycins A and B.

Two new 4'-acetylated analogs of chrysomycin were discovered during the screening for antitumor agents from the metabolites of actinomycetes. Their structures and physicochemical properties were determined by standard spectrometric analyses. Their cytotoxicities and antimicrobial activities were evaluated against a panel of cancer cell lines and microbes. While acetylation reinforced the cytotoxicity of chrysomycin B, it weakened the activity of chrysomycin A. Chrysomycin A and its acetylated analog showed high cytotoxicity toward most of the cancer cells with IC50s less than 10 ng ml-1. The 4'-acetyl-chrysomycin A was predominantly observed in nuclei at concentrations where the autofluorescence was observable. Chrysomycins were effective toward Gram-positive bacteria. The 4'-acetylated-chrysomycin A and B had MICs of 0.5-2 µg ml-1 and 2 to greater than 64 µg ml-1, respectively, toward Gram-positive bacteria including MRSA and VRE.

Novel approaches for identification of anti-tumor drugs and new bioactive compounds.

Thanks to the pioneering work done by Professor Hamao Umezawa, bioactive compounds have been used in treatment of several diseases including cancer. In this review, we discuss our work, which focuses on developing new candidates for anti-tumor drugs by screening for bioactive natural compounds in microbial cultures using unique experimental systems. We summarize our recent progress including the following: (1) small-molecule modulators of tumor-stromal cell interactions, (2) inhibitors of three-dimensional spheroid formation of cancer cells, (3) multi-cancer cell panel screening and (4) new experimental animal models for cancer metastasis.The Journal of Antibiotics advance online publication, 30 August 2017; doi:10.1038/ja.2017.97.

ATP Depletion Assay Led to the Isolation of New 36-Membered Polyol Macrolides Deplelides A and B from Streptomyces sp. MM581-NF15.

New 36-membered polyol macrolides deplelides A and B were isolated from the culture of Streptomyces MM581-NF15 by bioassay-guided fractionation using an ATP depletion assay. The planar structures of these novel compounds were identified by interpretation of the spectroscopic data (1D/2D NMR, MS, and IR). The relative stereochemistry was partially established using the universal NMR database method and J-based configuration analysis using 1H-1H and long-range 1H-13C coupling constants determined by 1H NMR or E.COSY and J-resolved HMBC analysis or another HMBC-based technique, respectively. The absolute stereochemistry was partially determined by a modified Mosher's method. These new compounds displayed highly potent ATP depletion activities (IC50 33 nM) and antiproliferative activities against several tumor cell lines, such as HGC-27 (IC50 47 nM).

Design, synthesis, and evaluation of DNA topoisomerase II-targeted nucleosides.

We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small structural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucleoside analogues containing TBS and 1,3-dithian moieties had potent and selective DNA topoisomerase II inhibitory activities. In further experiments, compound 25b having a beta configuration of the thymine moiety showed relatively strong growth inhibitory activity against cancer cell lines, and was more potent against all cancer cell lines than compound 26b, which carries a thymine moiety in the alpha configuration.