Yoshihito Ueno

SYNTHESIS OF ADENOPHOSTIN ANALOGUES LACKING THE ADENINE MOIETY AS NOVEL POTENT IP3 RECEPTOR LIGANDS : SOME STRUCTURAL REQUIREMENTS FOR THE SIGNIFICANT ACTIVITY OF ADENOPHOSTIN A

1-O-Tetrahydrofuranyl-α-d-glucopyranose derivatives 5−8 were designed and synthesized as novel IP3 receptor ligands. The glycosidation reactions between fluoroglycosyl donor 23 and tetrahydrofuran derivatives 11−14 as glycosyl acceptors selectively gave the corresponding α-glycosides, which were converted into the target compounds 5−8 via the introduction of phosphate groups using the phosphoramidite method. Among these compounds, 1-O-tetrahydrofuranyl-α-d-glucopyranose trisphosphate derivatives 5 and 8 significantly inhibited the binding of [3H] IP3 to IP3 receptor from porcine cerebella, with IC50 values of 25 and 27 nM, respectively, which were comparable to the affinity of IP3 itself.

A potent 2'-O-methylated RNA-based microRNA inhibitor with unique secondary structures.

MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of ‘Tough Decoy (TuD) RNA’ molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2′-O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization‐induced neurite outgrowth and the expression of growth‐associated protein‐43 (GAP‐43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP‐43 and synapsin I. These increases were suppressed by inhibition of Ca2+‐calmodulin‐dependent protein kinase II (CaMKII), PLD, or mitogen‐activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization‐induced neurite outgrowth, and the increase in GAP‐43 and synapsin I expression. Depolarization evoked a Ca2+ rise that activated various signaling enzymes and the cAMP response element‐binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl‐induced phosphorylation of proline‐rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal‐regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1‐butanol and transfection of PLD2 siRNA, whereas it was enhanced by over‐expression of wild‐type PLD2. Depolarization‐induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization‐induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP‐43 and synapsin I expression during depolarization‐induced neuronal differentiation in PC12 cells.

Diazirine-containing RNA photo-cross-linking probes for capturing microRNA targets.

Here, we report the applicability of diazirine-containing RNA photo-cross-linking probes for the identification of microRNA (miRNA) targets. The RNA cross-linking probes were synthesized by substituting the RNA nucleobases with nucleoside analogues such as 1-O-[3-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-β-d-ribofuranose or 1-O-[4-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-β-D-ribofuranose that carry aryl trifluoromethyl diazirine moieties. The probes were successfully cross-linked with synthetic RNAs containing the four natural nucleosides on the opposite site of the nucleoside analogues. Furthermore, it was found that miRNAs containing these analogues were effective in regulating the expression of their target genes. Thus, RNAs containing the nucleoside analogues are promising candidates as photo-cross-linking probes to identify the target mRNAs of miRNAs.

Chemical synthesis of cytidine-5′-monophosphono-N-acetylneuraminic acid (CMP-neu5ac)

Abstract CMP-Neu5AC was synthesized for the time from the cytidine 5′-phosphoramidite and the Neu5Ac derivative bearing allyl and allyloxycarbonyl groups as the protecting groups.

Functional Characterization of 2′,5′-Linked Oligoadenylate Binding Determinant of Human RNase L

RNase L is activated by the binding of unusual 2′,5′-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that π-π stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L·2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.

Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase

The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K(i)=0.148 mM and a mixed-type inhibitor with regard to ATP with measured K(i)=0.4 mM. The specificity constant (K(cat)/K(m)) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.

Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either NU or NUm and of DNA:RNA hybrid duplexes containing NUm. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to NU, NUm decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the NUm containing DNA:DNA duplex structure. The conformation of NUm, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of NUm to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.

Nucleosides and nucleotides. 161. Incorporation of 5-(N-aminoalkyl)carbamoyl-2′-deoxycytidines into oligodeoxyribonucleotides by a convenient post-synthetic modification method

Abstract 5-Trifluoroethoxycarbonyl-2′-deoxycytidine derivatives were synthesized and incorporated into oligonucleotides. The fully protected oligonucleotides were treated with diaminoethane or 1,6-diaminohexane to give oligonucleotides carrying amino-linkers, which were further derivatized with an intercalator. The properties of these oligonucleotides are described.

Nucleic acid probe containing fluorescent tricyclic base-linked acyclonucleoside for detection of single nucleotide polymorphisms

The development of a reliable and simple method for detecting single nucleotide polymorphisms (SNPs), common genetic variations in the human genome, is currently an important research area because SNPs are important for identifying disease-causing genes and for pharmacogenetic studies. Here, we developed a novel method for SNP detection. We designed and synthesized DNA probes containing a fluorescent tricyclic base-linked acyclonucleoside P. The type of nucleobases involved in the SNP sites in the DNA and RNA targets could be determined using four DNA probes containing P. Thus, this system would provide a novel and simple method for detecting SNPs in DNA and RNA targets.