Novel Benzo[B]Furans with Anti-Microtubule Activity Upregulate Expression of Apoptotic Genes and Arrest Leukemia Cells in G2/M Phase.

BACKGROUND: Novel derivatives of benzo[b]furan were found to be highly toxic towards human chronic myelogenous (K562), acute myelogenous (HL-60) and acute lymphoblastic (MOLT-4) leukemia cells. OBJECTIVE: The objective was the characterization of the biological activity of novel benzofurans (influence on apoptosis, mitogen-activated protein kinases and on the cell cycle). Cellular protein(s) targeted by test benzofurans and mechanism of action were identified. METHODS: The methods utilized in the study were chemical synthesis, fluorescence assays, flow cytometry, gene expression by DNA microarray and real-time RT-PCR, western blotting, cytotoxicity assays, pull-down assay, mass spectroscopy, in vitro polymerization of tubulin, molecular docking. RESULTS: 1,1'-[3-(bromomethyl)-5,6- dimethoxy-1-benzofuran-2,7-diyldiethanone (1) and methyl 4-bromo-6- (dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (2) induced apoptosis in K562 and MOLT-4 cells. The profiling of gene expression revealed that 1 and 2 increased the expression of proapoptotic genes involved in both receptor (TNFRSF 10A, TNFRSF 10B, CASP8) and mitochondrial (BAX, BID, NOXA, APAF1) pathways of apoptosis. Test benzo[b]furans activated c-Jun N-terminal kinase (JNK) and p38 kinase in K562 cells. Tubulin was identified as a protein target for benzo[b]furans in pull-down experiments with biotinylated 2. Test benzo[b]furans inhibited polymerization of tubulin monomers in vitro, decreased the level of cellular microtubules and arrested cells in a G2/M phase. Molecular docking suggests that benzo[b]furans 1 and 2 bind to tubulin via colchicine binding pocket and the complex is stabilized mainly by hydrophobic interactions. CONCLUSION: Novel benzo[b]furans with anti-microtubule activity were identified. They induce apoptosis in cancer cells and cause G2/M cell cycle arrest. Biological activity of 1 and 2 makes them potential lead compounds for development as anticancer drugs.

Cytochrome†c Catalyzes the Hydrogen Peroxide-Assisted Oxidative Desulfuration of 2-Thiouridines in Transfer RNAs.

The 5-substituted 2-thiouridines (R5S2Us) present in the first (wobble) position of the anticodon of transfer RNAs (tRNAs) contribute to accuracy in reading mRNA codons and tuning protein synthesis. Previously, we showed that, under oxidative stress conditions in vitro, R5S2Us were sensitive to hydrogen peroxide (H2 O2 ) and that their oxidative desulfuration produced 5-substituted uridines (R5Us) and 4-pyrimidinone nucleosides (R5H2Us) at a ratio that depended on the pH and an R5 substituent. Here, we demonstrate that the desulfuration of 2-thiouridines, either alone or within an RNA/tRNA chain, is catalyzed by cytochrome c (cyt c). Its kinetics are similar to those of Fenton-type catalytic 2-thiouridine (S2U) desulfuration. Cyt c/H2 O2 - and FeII -mediated reactions deliver predominantly 4-pyrimidinone nucleoside (H2U)-type products. The pathway of the cyt c/H2 O2 -peroxidase-mediated S2U→H2U transformation through uridine sulfenic (U-SOH), sulfinic (U-SO2 H), and sulfonic (U-SO3 H) intermediates is confirmed by LC-MS. The cyt c/H2 O2 -mediated oxidative damage of S2U-tRNA may have biological relevance through alteration of the cellular functions of transfer RNA.

Versatile Method for the Site-Specific Modification of DNA with Boron Clusters: Anti-Epidermal Growth Factor Receptor (EGFR) Antisense Oligonucleotide Case.

A general and convenient approach for the incorporation of different types of boron clusters into specific locations of the DNA-oligonucleotide chain based on the automated phosphoramidite method of oligonucleotide synthesis and post-synthetic "click chemistry" modification has been developed. Pronounced effects of boron-cluster modification on the physico- and biochemical properties of the antisense oligonucleotides were observed. The silencing activity of antisense oligonucleotides bearing a single boron cluster modification in the middle of the oligonucleotide chain was substantially higher than that of unmodified oligonucleotides. This finding may be of importance for the design of therapeutic nucleic acids with improved properties. The proposed synthetic methodology broadens the availability of nucleic acid-boron cluster conjugates and opens up new avenues for their potential practical use.

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted "click" conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400-2650 cm-1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 µM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

Reaction of S-geranyl-2-thiouracil modified oligonucleotides with alkyl amines leads to the N2-alkyl isocytosine derivatives.

S-Geranylated 2-thiouridines (geS2Us) are unique hydrophobic modified nucleosides identified very recently in bacterial tRNAs. Our study on the synthesis of geS2Ura-containing oligonucleotides (geS2U-RNA and geS2dU-DNA) revealed a fast substitution of the S-geranyl moiety by methylamine (frequently used in oligonucleotide deprotection procedures) or n-butylamine, providing the corresponding N2-alkyl isocytosine (R2isoCyt) derivatives. To retain the S-geranyl moiety in the DNA or RNA chains, the optimized deprotection protocol with 8 M ethanolic ammonia should be applied. The oligomers bearing the R2isoCyt heterocycle (R2isoC-RNA and R2isodC-DNA) are less hydrophobic than the corresponding S2U- and geS2U-modified oligomers, whereas, contrary to the previously reported data, geS2dU-DNA and geS2U-RNA exhibit significantly higher lipophilicity than the parent S2Ura-containing oligonucleotides. Thermodynamic studies revealed that: (a) both geS2Ura- and R2isoCyt-modified oligomers exhibit similar hybridization properties towards DNA and RNA templates, and (b) the R2isoCyt nucleobase preferentially hybridizes to guanine moiety in the DNA/DNA and RNA/RNA duplexes.

Synthesis, crystallization and preliminary crystallographic analysis of a 52-nucleotide DNA/2'-OMe-RNA oligomer mimicking 10-23 DNAzyme in the complex with a substrate.

A 52-nucleotide DNA/2'-OMe-RNA oligomer mimicking 10-23 DNAzyme in the complex with its substrate was synthesized, purified and crystallized by the hanging-drop method using 0.8 M sodium potassium tartrate as a precipitant. A data set to 1.21 Å resolution was collected from a monocrystal at 100 K using synchrotron radiation on a beamline BL14.1 at BESSY. The crystal belonged to the P21 group with unit-cell a = 49.42, b = 24.69, c = 50.23, ß = 118.48.

An efficient approach for conversion of 5-substituted 2-thiouridines built in RNA oligomers into corresponding desulfured 4-pyrimidinone products.

An efficient approach for the desulfuration of C5-substituted 2-thiouridines (R5S2U) bound in the RNA chain exclusively to 4-pyrimidinone nucleoside (R5H2U)-containing RNA products is proposed. This post-synthetic transformation avoids the preparation of a suitably protected H2U phosphoramidite, which otherwise would be necessary for solid-phase synthesis of the modified RNA. Optimization of the desulfuration, which included reaction stoichiometry, time and temperature, allowed to transform a set of ten R5S2U-RNAs into their R5H2U-RNA congeners in ca. 90% yield.

2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon-anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3'-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

siRNAs modified with boron cluster and their physicochemical and biological characterization.

RNA interference (RNAi) technology provides a powerful, yet selective, molecular tool to reduce the expression of genes in eukaryotic cells. Despite the success associated with the effective use of siRNA duplexes for gene silencing, there is a need to improve their properties. These properties, related mainly to migration through the cell membranes, stability of siRNA in vivo, and specificity of their silencing activity, can be improved by chemical modifications of siRNA backbone. In this study, we examined the physicochemical and biological properties of siRNA duplexes targeted against BACE1 gene modified at various positions with a lipophilic boron cluster (C2B10H11, CB). The lipophilicity and resistance to enzymatic degradation of the modified oligomers was higher than the unmodified counterparts. As measured in a dual fluorescence assay (BACE1-GFP/RFP), the carboranyl siRNAs (CB-siRNAs) were as active as the parent nonmodified duplexes and their toxicity toward HeLa cells was also similar. The helical structure of CB-siRNAs remained unchanged upon boron cluster introduction, as determined by CD and UV melting experiments.

The 2-thiouridine unit in the RNA strand is desulfured predominantly to 4-pyrimidinone nucleoside under in vitro oxidative stress conditions.

The 2-thiouridine (S2U) unit in the RNA strand is predominantly desulfured with H(2)O(2) to 4-pyrimidinone nucleoside (H2U). The resulting H2U-RNA exhibits significantly lower binding affinity to its complementary strand and in certain conditions undergoes strand scission. These results may explain the tRNA loss of biological function in oxidative stress conditions.

Longer 19-base pair short interfering RNA duplexes rather than shorter duplexes trigger RNA interference.

Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Recently, it was suggested that 16-bp siRNAs are effective RNAi triggers and superior to "classical" 19-bp siRNAs. This contradiction with generally accepted knowledge prompted us to reinvestigate this issue. Here, in a series of experiments performed with siRNA duplexes of various lengths (from 19 to 15 bp) designed to silence either overexpressed enhanced green fluorescent protein or endogenously expressed CDK9, we demonstrate that 19-bp siRNAs are more active silencers than shorter corresponding duplexes. The discrepancy between our results and those questioned appears to be due to different modes of shortening the duplex (either at the 3'-end or at the 5'-end, with respect to polarity of the guide strand). Importantly, duplexes with intact 5'-ends but shortened at their 3'-ends retain target site specificity, whereas those shortened at the 5'-end are complementary to different target sites located upstream.

A novel class of DNA analogs bearing 5'-C-phosphonothymidine units: synthesis and physicochemical and biochemical properties.

S(C) and R(C) diastereomers of 5'-C-(O,O-diethyl)-phosphonylthymidine ((R)T and (S)T) were used for the synthesis of the dimers T(R)T and T(S)T, respectively. These dimers were incorporated at selected sites in oligonucleotide constructs. Melting temperature (Tm) experiments demonstrated that relative to the unmodified oligodeoxyribonucleotide, the presence of the (R)T moiety reduced the thermal stability of the duplexes by approximately 5.0 degrees C per modification, whereas their (S)T counterparts only slightly destabilized the duplex structure (deltaTm < or = 1 degree C/modification). The stability of the triple-helical complexes containing one, two, or three modified thymidines is slightly higher than that of the parent complex. Nuclease resistance studies performed with snake venom phosphodiesterase, calf spleen phosphodiesterase, and 3'-exonuclease from human plasma showed that cleavage of the oligonucleotides at the site of the modification was completely suppressed regardless of the stereochemistry of the 5'-C-chiral center. The influence of the (R)T and (S)T modification in the recognition sequence of HindIII, EcoRI, and HpaI restriction endonucleases was also investigated. Although the catalytic activity of HindIII was not affected by the presence of the 5'-C-ethoxyphosphonyl modification, the activities of the two remaining restriction enzymes were partially suppressed depending on the site of modification or the stereochemistry of the modification or both ((R)T vs. (S)T).

Synthesis of dinucleoside (N3'-->MeP5') methanephosphonamidates.

[structure: see text] Three different approaches were used for the synthesis of dinucleoside methanephosphonamidates [3'-NH-P(O)(CH3)O-5'], starting from dichloromethylphosphine or dichloromethanephosphonate as the phosphorus-containing moiety. 5'-DMT-3'-amino-3'-deoxythymidine and N(4)-benzoyl-5'-DMT-3'-amino-2',3'-dideoxycytidine were used as the aminonucleoside precursors and the respective 3'-protected nucleosides (thymidine or N(4)-benzoyl-2'-deoxycytidine) as the 5'-hydroxyl reagents.