Preparation of Oligomeric 2′-Deoxy-5-Fluorouridylate of Defined Length and Backbone Composition: A Novel Pro-Drug form of the Potent Anti-Cancer Drug 2′-Deoxy-5-Fluorouridylate

Abstract

A recursive protection scheme for FdUMP is employed by synthesizing oligonucleotides consisting of only FdU. 3′-O-exonuclease action releases FdUMP and a shortened oligonucleotide from which further exonuclease action releases more FdUMP. Such oligonucleotides are more cytotoxic to H4IIe, mouse fibroblast, and metastatic mouse rumor cells than are equivalent concentrations of FdU monomer.     

Enhancing the Stability of Oligonucleotide Duplexes. The Effect of Adding 1-(2"-Deoxy-β-D-ribofuranosyl)-5-nitroindole

We studied the properties of DNA duplexes containing 5-nitroindole (N) in one of the chains. We synthesized 8-membered oligos with N at the 5" or at the 3" end: 5"-d(NXGACCGTC)-3" or 5"-d(GACCGTCXN)-3", where X is one of the four natural bases, making all four kinds of oligos with and without N. We also prepared 11-membered oligos complementary to the above octanucleotides: 5"-d(TGACGGTCYZT)-3" and 5"-d(TZYGACGGTCT)-3", where Y and Z are A, G, C, or T. The stability of duplexes obtained with these oligos was assessed by melting, and the thermodynamic parameters H, S, and T _m were calculated. Comparison of the melting curves for modified and nonmodified duplexes demonstrated that the presence of N at the 5" end of one chain raises the T _m by 6.6° on average; if N is at the 3" end of the same chain, the T _m increases by about 3°.     

Synthesis of a Novel C-Nucleoside, 2-Amino-7-(2-deoxy-β- D-erythro-pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one (2′-Deoxy-9-deazaguanosine)

Abstract

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2′-deoxyguanosine) was achieved starting from 2-amino-6-metnyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)- D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by ¹H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.     

A Novel Approach to Synthesis of 2′-Deoxy-β-D-Ribonucleosedes Via Transglycosylation of 6-Oxopurine Ribonucleosides

Abstract

A novel method of synthesis of 2′-deoxy-β-d-ribonucIeosides via transglycosylation of 6-oxopurine ribonucleosides is exemplified for conversion of inosine into 6-metylpurine 2′-deoxyriboside (5). The method offers high regio- and stereoselectivity as well as a good overall yield, and in these respects is superior to the fusion or anionic glycosylation procedures.

     

Conformationally Locked Nucleoside Analogs. Synthesis of 2′-Deoxy-2′-C, 4′-C-Bridged Bicyclic Nucleosides

Abstract

1-α-Methylarabinose was converted, in three steps, to 2-deoxy-2-methyleneribose derivative 3, which was subjected to hydroboration to give 2-α-hydroxymethyl derivative 4 exclusively. 4 was converted to 2,4-bis(hydroxymethyl)ribose derivative 6 in four steps. Mesylation, detritylation, and ring closure, followed by hydrolysis of the mesyl group at O5, gave 3,6-dioxabicyclo[3,2,1]octane derivative 8. After acetylation, 8 was coupled with silylated 6-chloropurine to give desired α- and β-bicyclic-sugar nucleosides.     

GL3, a Novel 4β-Anilino-4′-O-Demethyl-4-Desoxypodophyllotoxin Analog,Traps Topoisomerase II Cleavage Complexes and Exerts Anticancer Activities

A novel VP-16 derivative, 4β-[N -(4?-acetyloxyl-phenyl-1?-carbonyl)-4″-aminoanilino]-4′-O-demethyl-4-desoxypodophyllotoxin (GL3), displayed a wide range of cytotoxicity in a panel of human tumor cell lines, with half-maximal inhibitory concentration (IC₅₀) values ranging from 0.82 to 4.88 µM, much less than that of VP-16 (4.18–39.43 µM). Importantly, GL3 induces more significant apoptosis and cell cycle arrest than VP-16. The molecular and cellular machinery studies showed that GL3 functions as a topoisomerase II (Top 2) poison through direct binding to the enzyme, and the advanced cell-killing activities of GL3 were ascribed to its potent effects on trapping Top 2-DNA cleavage complex, Moreover, GL3-triggered DNA double-strand breaks and apoptotic cell death were in a Top 2-dependent manner, because the catalytic inhibitor aclarubicin attenuated these biologic consequences caused by Top 2 poisoning in GL3-treated cells. Taken together, among a series of 4β-anilino-4′-O-demethyl-4-desoxypodophyllotoxin analog, GL3 stood out by its improved anticancer activity and well-defined Top 2 poisoning mechanisms, which merited the potential value of GL3 as an anticancer lead compound/drug candidate deserving further development.     

9-Deoxy-Δ9-prostaglandin D2, a prostaglandin D2 derivative with potent antineoplastic and weak smooth muscle-contracting activities

We have recently described a potential antineoplastic effect of prostaglandin D₂ (PGD₂) (Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S., and Hayaishi, O. (1982) Biochem. Biophys. Res. Commun. $\text{105}$, 956–964). In the present study three analogues of PGD₂ were examined for their antineoplastic and other biological activities. 9-Deoxy-Δ⁹-PGD₂, a dehydrate of PGD₂, had about 3 times stronger growth inhibitory effect than PGD₂ on L1210 leukemia cultured cells. The IC₅₀ value of this compound was 0.5–1.0 μg/ml (2μ$\text{M}$). In contrast to PGD₂, this compound lacked a contracting activity on colonic smooth muscle and caused less diarrhea. The antiaggregatory action of the compound on blood platelet was about 10 times weaker than PGD₂. The growth inhibitory activity was also observed with cyclopentenone itself; the IC₅₀ value was about 100 μM. On the other hand, BW245C which had the antiaggregatory activity comparable with PGD₂ showed no growth inhibition. These results suggest that cyclopentenone structure is an essential moiety of prostaglandin derivaties for cell growth inhibition and that the growth inhibitory activity could be dissociated from other biological activities of prostaglandins.     

Synthesis of 9-(2-Deoxy-2-Fukuoro-β-D- Abinoruranosyl)Hypoxhine. The First Direct Introduction of A 2′-β-Fldoro Substituent in Ppepormed Purine Nucleosides. Studies Directed Toward the Synthesis of 2′-DEOXY-2′-Substituted Arabppmocebosides. 8.1

Abstract

The 3′, 5′-di-O-acetyl-, 3′-, 5′-di-O-balzyl-, 3′-O-acety -5-O-trityl- and 3′-, 5′ -di-O-trityl-2′-O-triflyl-1-benzylhnosine (8c, 15, 20C, and 27, respectively) were prepared and subjected to nucleophilic reaction with TASF. Thus, 3′, 5′-O-(1, 1, 3, 3-tetraisopropyldisiloxanyl)-1-benzylinosine (5c) was triflylated, desilylated, and then acetylated to give 8c. Also, 5c was converted into the 2′-O-tetrahydropyrnyl (W) derivative 11 which was desilylated and then benzylated to give 2′-O-tetrahydropyranyl-O^3′, O^5′, N¹-tribenzylinosine (13). Removal of the THP group from 13 followed by triflylation afforded 2′-O-triflyld-O^3′,O^5′ N¹-tribenzylinosine (15). 3′-O-Acetyl-2′ -O-triflyl-,O^5′,N¹-inosine (20) was prepared frmn 5′ -O-trityl-1-benzylhh (18c) by conversion into the 2′-, 3′-O-(di-n-butylstannylene) derivative which was treated with triflyl chloride and then acetylated. Treatment of 1-benzyl-inosine (4c) with trityl chloride in pyridine containing p-dimethylamino-pyridine afforded a mixture of 2′-, 5′- and 3′-, 5′-di-O-trityl-l-benzylinosine (25 and 26, respectively). These regioiscums were chrcanato-graphically separated. Triflylation of 26 gave 2′-o-triflyl-3′-, 5′-di-O-trityl-1-benzylhoshe (27).

The triflates 8c and 15 only afforded elhination products upon treatment with TASF. However, the trif late group in 20c and 27 was displaced by fluoride with fornation of the 2′-fluoro-arabino nucleosides, 21c and 28, in 10 and 30% yield, respectively. After deprotection of 28, 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)hypowntkine (1, F-ara-H) was obtained in good yield. The conformational influence of the sugar protecting groups on the rate of nucleophilic substitution against elimination is discussed.     

Rapid Emergence of Novel GII.4 Sub-Lineages Noroviruses Associated with Outbreaks in Huzhou,China, 2008–2012

Infection caused by noroviruses (NoVs) is one of the most important causes of acute gastroenteritis in humans worldwide. To gain insight into the epidemiology of and genetic variation in NoV strains, stool samples collected from 18 outbreaks of acute gastroenteritis in Huzhou, China, between January 2008 and December 2012 were analyzed. Samples were tested for NoVs by real-time RT-PCR. Partial sequences of the RNA- dependent RNA polymerase (RdRp) and capsid gene of the positive samples were amplified by RT-PCR, and the PCR products were sequenced and used for phylogenetic analysis. NoVs were found to be responsible of 88.8% of all nonbacterial acute gastroenteritis outbreaks in Huzhou over the last 5 years. Genogroup II outbreaks largely predominated and represented 93% of all outbreaks. A variety of genotypes were found among genogroups I and II, including GI.4, GI.8, GII.4, and GII.b. Moreover, phylogenetic analyses identified two recombinant genotypes (polymerase/capsid): GI.2/GI.6 and GII.e/GII.4 2012 Sydney. GII.4 was predominant and involved in 8/10 typed outbreaks. During the study period, GII.4 NoV variants 2006b, New Orleans 2009, and Sydney 2012 were identified. This is the first report of the detection of GII.4 New Orleans 2009 variant, GII.e/GII.4 Sydney 2012 recombinant in outbreaks of acute gastroenteritis in China.     

Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor,IRE1β

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1α and IRE1β, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1β senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1β clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1β complex is robustly increased upon ER stress. Moreover, unlike IRE1α, the luminal domain of IRE1β showed anti-aggregation activity in vitro, and IRE1β was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1β, while IRE1α was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1α, the luminal event mediated by IRE1β involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.     

α4β2-Nicotinic Receptor Binding with 5-IA in Alzheimer’s Disease: Methods of Scan Analysis

Five patients with Alzheimer’s disease and five healthy volunteers were examined by SPECT with the nicotinic receptor ligand ¹²³I-5-IA-85380. Patients were scanned before and after 6 weeks of treatment with donepezil. Quantification by regions of interest was reliable and the optimal normalisation procedure used cerebellar ratios. We found relative reductions in 5-IA binding capacity in patients in thalamus, frontal and central regions of interest of approximately one standard deviation unit (Cohen’s d = 1). Reductions in binding after treatment with the acetylcholinesterase inhibitor donepezil of the same magnitude occurred in the brain stem. The study was clearly too small to confirm group differences, but it suggests that 5-IA can be used to examine both group differences and treatment effects in patients with Alzheimer’s disease. Special issue article in honor of George Fink.     

Characterization of a novel α4/4-conotoxin,Qcl.2, from vermivorous Conus quercinus

As part of continuing studies of the identification of gene organization and cloning of novel α-conotoxins, the first α4/4-conotoxin identified in a vermivorous Conus species, designated Qcl.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds （Ⅰ-Ⅲ, Ⅱ-Ⅳ connectivity） in a native globular configuration. The mature peptide of Qcl.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qcl.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qcl.2 was shown to selectively inhibit both rat neuronal α3β2 and α3β4 subtypes of nicotinic acetylcholine receptors with low potency. A block of -63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized α-conotoxin members, the unusual structural features in Qcl.2 that confer to its receptor recognition profile are addressed.     

Synthesis of 4-Amino-8-(2, 2-Difluoro-2-Deoxy-β-D-Ribo Furanosyl Amino)Pyrimido [5, 4-D]Pyrimidine (dFARPP). Stability and Cellular Cytotoxicity

Abstract

The synthesis of β-dFARPP was accomplished by Mitsunobu coupling of 6-cyanopurine with 2-deoxy-2, 2-difluororibose followed by purine ring-expansion under the action of methanolic ammonia. The title compound inhibited the growth of proliferating human leukemic CCRF-CEM cells at 6.1μg/ml but had no effect on plaque formation by a variety of DNA and RNA viruses.     

miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP,CK2-α, CCNJ,and MEGF9

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3’-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40–56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.     

Synthesis of 2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide (3′-Deoxytiazofurin)

Abstract

2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide was synthesized in four steps from its β-D-ribofuranosyl nucleoside precursor.     

Proinflammatory Secreted Phospholipase A2 Type IIA (sPLA-IIA) Induces Integrin Activation through Direct Binding to a Newly Identified Binding Site (Site 2) in Integrins αvβ3, α4β1, and α5β1

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.